Scientific Committee on Consumer Safety...

SCCS/1516/13 Revision of 22 April 2014

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Scientific Committee on Consumer Safety 10

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SCCS 12

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OPINION ON 22

Titanium Dioxide (nano form) 23

COLIPA n° S75 24

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31 The SCCS adopted this opinion by written procedure on 22 July 2013 32

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SCCS/1516/13

Revision of the opinion on Titanium Dioxide, nano form

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About the Scientific Committees 1

Three independent non-food Scientific Committees provide the Commission with the 2

scientific advice it needs when preparing policy and proposals relating to consumer safety, 3 public health and the environment. The Committees also draw the Commission's attention 4

to the new or emerging problems which may pose an actual or potential threat. 5 They are: the Scientific Committee on Consumer Safety (SCCS), the Scientific Committee 6

on Health and Environmental Risks (SCHER) and the Scientific Committee on Emerging and 7 Newly Identified Health Risks (SCENIHR) and are made up of external experts. 8

In addition, the Commission relies upon the work of the European Food Safety Authority 9 (EFSA), the European Medicines Agency (EMA), the European Centre for Disease prevention 10

and Control (ECDC) and the European Chemicals Agency (ECHA). 11

SCCS 12 The Committee shall provide opinions on questions concerning all types of health and safety 13

risks (notably chemical, biological, mechanical and other physical risks) of non-food 14

consumer products (for example: cosmetic products and their ingredients, toys, textiles, 15 clothing, personal care and household products such as detergents, etc.) and services (for 16

example: tattooing, artificial sun tanning, etc.). 17 18

19 Scientific Committee members 20

Ulrike Bernauer, Qasim Chaudhry, Pieter-Jan Coenraads, Gisela Degen, Maria Dusinska, 21 David Gawkrodger, Werner Lilienblum, Andreas Luch, Elsa Nielsen, Thomas Platzek, Suresh 22

Chandra Rastogi, Christophe Rousselle, Jan van Benthem. 23

24 Contact 25

European Commission 26 Health & Consumers 27

Directorate C: Public Health 28 Unit C2 – Health Information/ Secretariat of the Scientific Committee 29

Office: HTC 03/073 L-2920 Luxembourg 30 [email protected] 31

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© European Union, 2013 35

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ISSN 1831-4767 ISBN 978-92-79-30114-8 37

Doi: 10.2772/70108 ND-AQ-13-007-EN-N 38

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The opinions of the Scientific Committees present the views of the independent scientists 40

who are members of the committees. They do not necessarily reflect the views of the 41

European Commission. The opinions are published by the European Commission in their 42 original language only. 43

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http://ec.europa.eu/health/scientific_committees/consumer_safety/index_en.htm 45

mailto:[email protected]

http://ec.europa.eu/health/scientific_committees/consumer_safety/index_en.htm

SCCS/1516/13


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1

ACKNOWLEDGMENTS 2

3 SCCS Members 4

Dr. Q. Chaudhry (chairman and rapporteur) 5 Dr. U. Bernauer 6

Dr. W. Lilienblum 7 Dr. S. Ch. Rastogi 8

Prof. Th. Platzek 9 Dr. J. van Benthem 10

Dr. M. Dusinska 11

SCENIHR members 12 Prof. P. H. M. Hoet 13

External experts 14 Dr. W. Kreyling 15

Dr. R. Schins 16 Dr. W. H. de Jong 17

Dr. T. Jung 18 Prof. T. Sanner 19

Dr. J. van Engelen 20

Dr. O. Bussolati 21 Prof. M-P Vinardell 22

Dr. Sh. Doak 23

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For the revision 25 SCCS members 26

Dr. U. Bernauer 27 Dr. Q. Chaudhry (chairman and rapporteur) 28

Dr. M. Dusinska 29

Dr. W. Lilienblum 30 Prof. Th. Platzek 31

Dr. S. Ch. Rastogi 32 Dr. J. van Benthem 33

SCENIHR members 34 Prof. P. H. M. Hoet 35

Dr. K. Rydzynski 36 External experts 37

Dr. O. Bussolati 38

Dr. W. H. de Jong 39 Dr. S. H. Doak 40

Prof. Th. Jung 41 Prof. T. Sanner 42

Dr. N. von Götz 43 44

This opinion has been subject to a commenting period of six weeks after its initial 45 publication. Comments received during this time have been considered by the SCCS and 46

discussed in the subsequent plenary meeting. Where appropriate, the text of the relevant 47

sections of the opinion has been modified or explanations have been added. In the cases 48 where the SCCS after consideration and discussion of the comments, has decided to 49

maintain its initial views, the opinion (or the section concerned) has remained unchanged. 50 Revised opinions carry the date of revision. 51

52 Keywords: SCCS, scientific opinion, UV filter, titanium dioxide (nano form), SCCS/1516/13, 53

directive 76/768/ECC, CAS number: 13463-67-7, EC: 236-675-5 54 55

Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), Opinion on 56

titanium dioxide (nano form), 22 July 2013, revision of 22 April 2014. 57

SCCS/1516/13


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TABLE OF CONTENTS 1

1. BACKGROUND ............................................................................................. 6 2

1.1 TERMS OF REFERENCE ......................................................................... 6 3 1.2 OPINION ............................................................................................ 7 4

1.3 Chemical and Physical Specifications ....................................................... 7 5 1.3.1 Chemical identity .................................................................... 7 6

1.3.1.1 Primary name and/or INCI name ............................................... 7 7 1.3.1.2 Chemical names ...................................................................... 7 8

1.3.1.3 Trade names and abbreviations ................................................. 7 9 1.3.1.4 CAS / EC number..................................................................... 7 10

1.3.1.5 Structural formula ................................................................... 7 11

1.3.1.6 Empirical formula .................................................................... 7 12 1.3.2 Physical form ......................................................................... 7 13

1.3.3 Molecular weight .................................................................. 10 14 1.3.4 Purity, composition and substance codes .................................. 10 15

1.3.5 Impurities / accompanying contaminants ................................. 10 16 1.3.6 Solubility ............................................................................. 10 17

1.3.7 Partition coefficient (Log Pow) ................................................ 10 18 1.3.8 Additional physical and chemical specifications .......................... 10 19

1.3.9 Droplet size in formulation ..................................................... 12 20

1.3.10 Particle size ......................................................................... 13 21 1.3.11 Microscopy .......................................................................... 14 22

1.3.12 Homogeneity and stability ...................................................... 15 23 1.4 Function and uses .............................................................................. 18 24

1.5 Toxicological Evaluation ...................................................................... 18 25 1.5.1 Acute toxicity ....................................................................... 18 26

1.5.1.1 Acute oral toxicity .................................................................. 18 27 1.5.1.2 Acute dermal toxicity ............................................................. 20 28

1.5.1.3 Acute inhalation toxicity ......................................................... 22 29

1.5.2 Irritation and corrosivity ........................................................ 25 30 1.5.2.1 Skin irritation ........................................................................ 25 31

1.5.2.2 Mucous membrane irritation .................................................... 29 32 1.5.3 Skin sensitisation .................................................................. 31 33

1.5.4 Dermal / percutaneous absorption........................................... 33 34 1.5.5 Repeated dose toxicity .......................................................... 59 35

1.5.5.1 Repeated Dose (30 days) oral toxicity ...................................... 59 36 1.5.5.2 Sub-chronic (90 days) toxicity (oral, dermal) ............................. 59 37

1.5.5.3 Chronic (> 12 months) toxicity ................................................ 61 38

1.5.6 Mutagenicity / Genotoxicity .................................................... 61 39 1.5.6.1 Mutagenicity / Genotoxicity in vitro .......................................... 62 40

1.5.6.2 Mutagenicity/Genotoxicity in vivo ............................................. 73 41 1.5.7 Carcinogenicity ..................................................................... 77 42

1.5.8 Photo-carcinogenicity ............................................................ 84 43 1.5.9 Reproductive toxicity ............................................................. 85 44

1.5.9.1 Two generation reproduction toxicity ........................................ 87 45 1.5.9.2 Teratogenicity ....................................................................... 87 46

1.5.10 Toxicokinetics ...................................................................... 87 47

1.5.11 Photo-induced toxicity ........................................................... 89 48 1.5.11.1 Phototoxicity / photoirritation and photosensitisation ................ 89 49

1.5.11.2 Phototoxicity / photomutagenicity / photoclastogenicity ............. 92 50 1.5.12 Human data ......................................................................... 93 51

1.5.13 Special investigations ............................................................ 94 52 1.5.14 Human safety evaluation (including calculation of MoS) .............. 94 53

1.5.15 Discussion ........................................................................... 94 54 2. CONCLUSIONS .......................................................................................... 99 55

3. MINORITY OPINION .................................................................................. 102 56

4. REFERENCES ........................................................................................... 102 57 58

SCCS/1516/13


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1

1. BACKGROUND 2

3 The first scientific opinion on the safe use of titanium dioxide as a UV-filter at a maximum 4

concentration of 25% in cosmetic products was adopted 24 October 2000 by the SCCNFP 5 (SCCNFP/0005/98). 6

However, a review of the substance in its nanoform is deemed necessary according to the 7 opinion on Safety of Nanomaterials in Cosmetic Products adopted on 18 December 2007 8

(SCCP/1147/07), where it is stated that: 9 10

"The SCCNFP opinion from 2000 (SCCNFP/0005/98) is on micro-crystalline preparations of 11

TiO2 and preparations of coarse particles. However, since this opinion, new scientific data 12 on nanosized particles including, TiO2 has become available. Therefore, the SCCP considers 13

it necessary to review the safety of nanosized TiO2 in the light of recent information. Also, a 14 safety assessment of nanosized TiO2, taking into account abnormal skin conditions and the 15

possible impact of mechanical effects on skin penetration needs to be undertaken". 16 17

Supplementary information on nanosized Titanium dioxide was submitted following a 18 meeting with stakeholders on 1 October 2008, where data requirements were agreed. 19

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Titanium Dioxide is currently regulated - irrespectively of its form - as a UV-filter in a 21 concentration up to 25% in cosmetic products in Annex VII, entry 27 of the Cosmetics 22

Directive. 23 24

1.1 TERMS OF REFERENCE 25

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1. Does SCCS consider that use of titanium dioxide in its nanoform as a UV-filter 27

in cosmetic products in a concentration up to maximum 25.0 % is safe for the 28

consumers taken into account the scientific data provided? 29

2. In order for the COM to differentiate in the regulation between materials in its 30

nanoform and its non-nano form, can the SCCS give quantitative and 31 qualitative guidance on how this differentiation should be given based on the 32

particle size distribution or other parameters? 33

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SCCS/1516/13


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1.2 OPINION 1

2

1.3 Chemical and Physical Specifications 3

1.3.1 Chemical identity 4

Titanium Dioxide 5

1.3.1.1 Primary name and/or INCI name 6

Titanium Dioxide 7

1.3.1.2 Chemical names 8

Titanium Dioxide 9

1.3.1.3 Trade names and abbreviations 10

COLIPA No. S75 11

1.3.1.4 CAS / EC number 12

13 CAS number: 13463-67-7 14

15 EC: 236-675-5 16

17 Other registry numbers: 100292-32-8; 101239-53-6; 1025343-79-6; 116788-85-3; 12000-18

59-8; 1205638-49-8; 1236143-41-1; 12701-76-7; 12767-65-6; 12789-63-8; 1309-63-3; 19

1344-29-2; 1377807-26-5; 1393678-13-1; 1400974-17-5; 158518-86-6; 185323-71-1; 20 185828-91-5; 188357-76-8; 188357-79-1; 195740-11-5; 221548-98-7; 224963-00-2; 21

246178-32-5; 252962-41-7; 37230-92-5; 37230-94-7; 37230-95-8; 37230-96-9; 39320-22 58-6; 39360-64-0; 39379-02-7; 416845-43-7; 494848-07-6; 494848-23-6; 494851-77-3; 23

494851-98-8; 52624-13-2; 55068-84-3; 55068-85-4; 552316-51-5; 62338-64-1; 767341-24 00-4; 859528-12-4; 861455-28-9; 861455-30-3; 866531-40-0; 97929-50-5; 98084-96-9. 25

[Source: ChemIdPlus] 26

1.3.1.5 Structural formula 27

TiO2 28

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1.3.1.6 Empirical formula 30

Formula: TiO2 31 32

1.3.2 Physical form 33

Titanium Dioxide (TiO2¸ COLIPA No. S75, CAS No. 13463-67-7) is described as a solid, 34

white, odourless powder. The TiO2 materials used in sunscreen products are reported to be 35 composed of two crystalline types: rutile and anatase or a mixture of the two. The different 36

materials included in the dossier have been reported to be needle, spherical, or lanceolate 37

(longer than wide) in shape. The primary particle size of the TiO2 nanomaterials has been 38 reported to range from around 20 to 100 nm. 39

40 Nanoparticles are generally known to have a tendency to stick together to form 41

agglomerates and/or aggregates, and it is claimed by the Applicant that, in sunscreen 42 products, TiO2 is not present in the form of primary nanoparticles but as aggregates of a 43

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size between 30 nm to >150 nm. These aggregates are claimed to be formed during the 1 manufacturing process. 2

3 Fifteen (15) TiO2 nanomaterials have been presented in the submission for evaluation. They 4

include uncoated as well as surface-coated nanomaterials with various organic and inorganic 5 coating materials. A range of coating materials has been used which include hydrophilic, 6

hydrophobic and amphiphilic materials, such as alumina/silica, methicone/silica, aluminium 7 hydroxide and dimethicone/methicone copolymer, trimethyloctylsilane, alumina/silicone and 8

alumina/silica/silicone, dimethicone, simethicone, stearic acid, glycerol, 9 dimethoxydiphenylsilane, triethoxycaprylylsilane (Table 1). 10

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The coating materials have been stated by the Applicant to be those that are common 12 cosmetic ingredients. The purpose of coatings has been stated to include improvement of 13

the dispersion of TiO2 nanomaterials within the cosmetic formulation, inhibiting or 14 controlling photoactivity, and improving compatibility with other ingredients in sunscreen 15

formulations. The coatings applied to nanoparticle surface are also stated to be not UV 16 absorbers themselves. 17

18 SCCS Comment 19

For this opinion, the trade names of the nanomaterials under assessment have been coded 20

by the SCCS and are referred to by the relevant codes. 21

It has been stated by the Applicant that ‘[the stability of coating] is certainly less relevant 22

from a human-safety aspect, especially since materials used as coating agents for TiO2 may 23 be present as constitutive ingredients of the same cosmetic product’. This may be true for 24

some materials, but it also needs to be considered that a range of materials has been used 25 for coating the TiO2 nanomaterials under current assessment. Some of these materials have 26

been used in a substantially high coating to nanomaterial ratios (e.g. 16% alumina). 27 Although a few studies showing coating stability have been provided, it is important to know 28

whether this, for example, could lead to the release of aluminium ions from alumina that 29

may be present after the coating process and which may dissolve in the final formulation. 30 Thus, where appropriate, safety of the coating materials should also be considered in their 31

own right because any significant dissolution of a coating component, such as alumina, may 32 require a separate safety assessment. 33

34 Three studies have been provided (submission II – Ref 62 and 63, and Submission III – Ref 35

68) to indicate that the coatings (e.g. silica/alumina) are stable in formulation, as well as 36 under different conditions of pH, temperature, shear force, etc. However, from the other 37

physicochemical data provided, it is less clear how stable the coatings are in final 38

formulations. The photocatalytic activity data, measured in formulations, indicate that either 39 some of the materials were not completely coated, or some of the coatings were not stable 40

in the formulations. 41 42

Despite the fact that the materials used as coatings to TiO2 nanomaterials have a wide 43 diversity, and some of them have been used in substantially high proportions (e.g. 16% 44

alumina), putative exposure to the coating materials has not been considered in the 45 assessment. Although a few studies showing coating stability have been provided, it is 46

important to know the concentration of any dissolved coating materials, e.g. aluminium 47

ions, in the final formulation. For example, in a recent study, Virkutyte et al (2012) found 48 that chlorine in swimming pools could potentially strip the coating from titanium dioxide 49

nanoparticles in sunscreens. The study, however, relates to a specific use scenario – i.e. 50 where TiO2 nanomaterials are coated with aluminium hydroxide (Al(OH)3), and the product 51

is used in chlorinated water (e.g. in a swimming pool). It is also likely that the coating-52 stripped nanoparticles will be washed off the skin during swimming or bathing after 53

swimming. Although this specific type of coating/use scenario relates more to risk 54 management than risk assessment, any significant dissolution of some coating materials 55

(e.g. alumina) may require a separate safety assessment for the uncoated nanomaterial as 56

well as the coating material. 57

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In view of this, the SCCS has only recommended the types of coatings covered in this 1 opinion. Other cosmetic ingredients applied as stable coatings on TiO2 nanomaterials can 2

also be used, provided that they can be demonstrated to the SCCS to be safe and the 3 coatings do not affect the particle properties related to behaviour and/or effects, compared 4

to the nanomaterials covered in this opinion. 5 6

Table-1: Form and composition TiO2 nanomaterials * 7 8

Materialcode

TiO2 crystaline form

Coating material Doping material

Form Bulk density (g/cm3)

VSSA (m2 cm-3)

S75-A > 99.5% Rutile

6% silica, 16% alumina None Oil dispersion

0.35 460

S75-B > 99.5% Rutile

6% silica, 16% alumina None Aqueous dispersion

0.35 460

S75-C > 99.5% Rutile

7.5% alumina, 9,5% aluminium stearate

None Oil dispersion

0.31 220

S75-D > 99.5% Rutile

10% alumina, 13.5% stearate

None Oil dispersion

0.58 300

S75-E > 99.5% Rutile

10% alumina, 13.5% stearate

None Aqueous dispersion

0.58 300

S75-F Anatase 85%, Rutile 15%

7.5% trimethoxycaprylylsilane

None Hydrophobic powder

0.2 192

S75-G Anatase 85%, Rutile 15%

None None Hydrophilic powder

0.13 213

S75-H > 99,5% Rutile

6% alumina, 1% glycerin

None Hydrophilic powder

0.31 260

S75-I > 99,5% Rutile

7% alumina 10% stearic acid


0.28 300

S75-J > 99,5% Rutile

6% alumina 1% dimethicone


0.31 260

S75-K > 94% Rutile 6-8% aluminium hydroxide, 3.5-4.5% dimethicone/methicone copolymer


0.12-0.28 426

S75-L > 94% Rutile 6.5-8.5% hydrated silica, 2.5-4.5% aluminium hydroxide, 4.5-6.5% dimethicone/methicone copolymer


0.07-0.2 426

S75-M > 98% Rutile, <2% anatase

17% silica None Hydrophilic powder

0.09 260

S75-N > 95% Rutile, <5% anatase

Alumina 10% simethicone 2%

1000 ppm Fe

Amphiphilic powder

0.16 400

S75-O 100% Anatase

Simethicone 5% None Hydrophobic powder

0.75 400

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* Regarding purity/impurity all materials are claimed by the applicant to conform with USP 10 35 requirements: TiO2 (99.0-100.5%), Loss on Ignition (≤ 13%), Water-soluble substances 11

(≤ 0.25%), Acid-soluble substances (≤ 0.5%), Arsenic (≤ 1 ppm), Residual Solvents (No 12 solvents used), 13

and FDA requirements: Lead (HCl-soluble) (≤ 10 ppm), Antimony (HCl-soluble) (≤ 2 ppm), 14 Mercury (≤ 1 ppm). 15

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For purity/impurity, all materials were tested as uncoated and untreated material. 1 2

SCCS comment 3 Analytical data on purity and impurities were not submitted, purity was only referred to USP 4

and FDA requirements. Analytical data on purity and impurities of each nanomaterial should 5 be provided. 6

1.3.3 Molecular weight 7

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Molecular weight of TiO2: 79.9 g/mol. 9 10

1.3.4 Purity, composition and substance codes 11

According to the Applicant, the TiO2 nanomaterials have been produced according to USP 12 31 specifications, in high purity, with concentration of the active material ≥99.0 %. It is 13

also stated that the materials do not contain heavy metals (e.g., Hg, Cd, Pb, As or Sb) 14 beyond the generally accepted limits. 15

16 SCCS Comments 17

The nanomaterials included in the submission have been stated to be manufactured 18 according to USP-31 specifications, with no heavy metals beyond the ‘generally accepted 19

limits’. The Applicant should provide the contents of heavy metals, such as Hg, Cd, Pb, As 20

and Sb, which are considered ‘acceptable’ under USP-31, as they may or may not be 21 considered acceptable under the EU regulations. In addition, impurities of well-known 22

metallic contact allergens, such as Cr, Co, Ni, should also be reported. 23

Purity/impurity has been referred to USP-35 in the additional information provided by the 24

applicant. USP-31 is an earlier edition of USP-35. 25

26

1.3.5 Impurities / accompanying contaminants 27

See SCCS comment under 1.3.2 28

29

1.3.6 Solubility 30

TiO2 is insoluble in water and organic solvents. It also has a very low dissociation constant 31

in water and aqueous systems, and thus can in practice be considered as insoluble also 32 under the physiological conditions. 33

(Numerous references in open literature) 34 35

1.3.7 Partition coefficient (Log Pow) 36

Log Pow: Not applicable for uncoated TiO2. 37

(Reference: 137) 38

39 SCCS Comment 40

A method to determine partition coefficient of nano particles coated with organic materials 41 is not yet available. However, distribution of TiO2 nanomaterials coated with organic 42

substances between polar and non polar phases should be described. 43 44

1.3.8 Additional physical and chemical specifications 45

46

Melting point: Not provided 47

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Boiling point: Not applicable 1 Flash point: Not applicable 2

Vapour pressure: Not applicable 3 Density: The Tap Density of the titanium dioxide powders was 4

measured according to DIN ISO 787/11 (Table 1) 5 Viscosity: Not provided 6

pKa: Not applicable for uncoated TiO2 7 Refractive index: Not provided 8

UV_Vis spectrum (200-800 nm): UV data only (see Table 3) 9 10

SCCS Comment 11

The dissociation kinetics of the materials in acidic media can be potentially modified by 12 certain coatings. However, considering the physicochemical properties of TiO2, it is agreed 13

that, for TiO2 nanomaterials, coatings are unlikely by definition to change the dissociation 14 constant of TiO2 in water. 15

16 Table-2: Physicochemical properties of TiO2 nanomaterials 17

18 Material code

Crystal size

Aspect ratio

UV Absorption (Extinction coefficient)

Zeta potential

Photo-catalytic activity*

Photo-stability

Coating stability

(XRD) (L /W) E308 E360 E400 (IEP) ∆E % to Reference

S75-A 15 3.8 44 20 11 7 3 9 Photo-stable

Stable

S75-B 15 3.8 51 22 12 N/A 3 9 Photo-stable

Stable

S75-C 15 3.7 54 16 7 N/A 7.8 23 Photo-stable

Stable

S75-D 9 4.5 48 7 3 N/A 7.2 21 Photo-stable

Stable

S75-E 9 4.5 50 10 4 N/A 7.2 21 Photo-stable

Stable

S75-F 21 1.2 45 15 8 N/A 11.8 35 Photo-stable

Stable

S75-G 21 1.2 38 16 9 7 25.1 74 Photo-stable

NA

S75-H 21 1.7 30 17 9 7 0.3 1 Photo-stable

Stable

S75-I 15 3.2 38 14 6 N/A 0.8 2 Photo-stable

Stable

S75-J 21 1.5 36 16 9 N/A 0.6 2 Photo-stable

Stable

S75-K 15 3.9 60 12 1 N/A 2.3 7 Photo-stable

Stable

S75-L 15 4.3 55 14 2 N/A 0.8 2 Photo-stable

Stable

S75-M 20 2.6 26 12 5 2 0.6 2 Photo-stable

Stable

S75-N 13 4.1 45 13 5 9 0.7 2 Photo-stable

Stable

S75-O 18 1.2 20 8 5 N/A 15.7 46 Photo-stable

Stable

19 * Photocatalytic activity 5% TiO2 formulation irradiated in a Suntest CPS+ solar simulator 20 for 30 minutes at 300 W/m^2. Sample measured before and after using a colourimeter. 21

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Calculation ∆E = [(∆L*)^2+(∆a*)^2 + (∆b*)^2]^1/2; Reference uncoated TiO2 ∆E = 34. 1 See Egerton et al. (2007) for more details on the method. 2

3 SCCS Comment 4

The photoreactivity of a chemical is generally determined in terms of degradation of an 5 organic substance (e.g. iso-propanol, propanone, salicylic acid, or an organic dye such as 6

methylene blue) on exposure to UV irradiation. Regarding measurement of photocatalytic 7 activity of nanomaterials, the OECD guidance (2010) provides further information and also 8

cites the methods described in ISO TC 206/WG37 (Fine ceramics – Test methods for 9 photocatalytic material). 10

In regard to the TiO2 nanomaterials under evaluation, the SCCS accepted the applicant’s 11

provided data from a different method used for measuring photocatalytic activity. The 12 method, which is described by Egerton et al. (2007), is based on photogreying of the TiO2 13

material on exposure to UV irradiation. Although the test is based on a non-standard 14 method, the SCCS accepted the data in view of the published work by Egerton et al. (2007), 15

which indicates measurable photogreying of TiO2 nanomaterials upon UV irradiation. As 16 such, the method will not be applicable to other nanomaterials because they may not turn 17

grey on exposure to UV irradiation, and/or may already have a colour. 18 Nanomaterials used in cosmetic products should ideally be non photocatalytic. However, in 19

view of measurement uncertainties, the SCCS has considered acceptable an arbitrary level 20

of up to 10% photocatalytic activity of a coated or doped nanomaterial, measured in terms 21 of % to a reference standard (which is uncoated/undoped form of the same nanomaterial). 22

23

1.3.9 Droplet size in formulation 24

25 According to the information provided by the Applicant, sunscreen spray products containing 26

nano-sized TiO2 are available on the EU market. These spray products are formulated with 27 non-volatile ingredients in pump sprays (without propellant gas) to generate minimal 28

aerosol cloud. It is stated that these products comply with current standards and 29

requirements in terms of droplet size, Mass Median Aerodynamic Diameter (MMAD) of at 30 least 30 μm, with no more than 1% of the droplets having an aerodynamic diameter of 10 31

μm or less. The Applicant has quoted the Technical Guidance Document on Risk Assessment 32 of the European Chemical Bureau (2003), which considers aerosols with an MMAD >10-15 33

μm as not respirable for humans because of deposition mainly in the upper regions of the 34 lungs (Reference 148). It is also quoted that the U.S. Silicones Environmental, Health and 35

Safety Council (2001) suggests that a consumer aerosol application for any silicone-based 36 material, regardless of the method of aerosol generation, should have particle size MMAD at 37

least 30 μm, with no more than 1% of the particles having an aerodynamic diameter of 10 38

μm or less (Reference 203). The Applicant has provided droplet size distribution 39 measurements for a few sprayable products. The technique used for droplet size 40

measurement was based on Laser Diffraction by Malvern method. 41 42

SCCS Comments 43

- The trade name of one sprayable product suggests that it may be for use by children. 44

- The droplet size of an aerosolised formulation would affect the entry and uptake of 45

nanomaterial in the lung. It is therefore noteworthy that whilst droplet size would 46

depend on nebulizer/ matrix, it may change due to evaporation/sublimation of the fluid 47 used in the emulsion. Thus, the characteristic dimension of a nanomaterial contained in 48

the formulation would have little relevance to the droplet size, which is typically much 49

larger (tens of micron). 50

- Although the measurement results indicate that droplet sizes were largely above the 51

respirable range (>10 µm), and only 0.24 to 0.37% of the droplets were in the size 52

range below 20 µm, it should be noted that even a low fraction based on droplet weight 53 is still relevant because it will contain a large number of nanoparticles. The possibility of 54

droplets drying and becoming smaller in size following spraying, and the possible lung 55

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exposure to dried residual particles after inhalation also needs to be taken into account. 1 The measurement of the droplet size distribution therefore needs to be complemented 2

by measurements of the size distribution of the dried residual aerosol particles as well, if 3 they can dry on the timescale in a practical use scenario. 4

- The size distribution of the droplets and dried droplets/ particles should be presented as 5

number size distribution. 6

1.3.10 Particle size 7

8 Table 3: Particle size of TiO2 nanomaterials 9

Material code

Particle Size Distribution*

Lower Cut Off level (nm) Volume weighted median, X50,3 (nm)

Number weighted median, X50,0

(nm)

CPS LUMi-sizer DLS

Average** CPS

LUMi-sizer DLS

Average** CPS

LUMi-sizer DLS

Average**

S75-A 20 33 35 29 53 71 111 78 37 48 79 55

S75-B 28 34 47 36 68 76 145 96 47 56 105 69

S75-C 20 25 26 24 52 49 78 60 39 48 59 49

S75-D 17 23 15 18 35 44 56 45 28 34 34 32

S75-E 21 27 41 30 45 51 104 67 37 42 81 53

S75-F 35 49 63 49 75 92 139 102 55 70 115 80

S75-G 25 58 54 46 77 99 129 102 45 79 102 75

S75-H 29 63 41 44 71 120 112 101 50 79 82 70

S75-I 22 58 41 40 73 107 140 107 40 76 103 73

S75-J 33 52 35 40 71 103 125 100 48 69 85 67

S75-K 26 34 30 30 48 52 75 58 41 44 58 48

S75-L 33 37 41 37 56 64 103 74 46 53 80 60

S75-M 42 75 73 63 119 124 173 139 75 99 133 102

S75-N 21 37 26 28 51 61 91 68 41 51 65 52

S75-O 24 71 47 47 354 653 146 384 33 87 85 68

10

* The particle size distribution was measured by three different methods - Differential 11 Sedimentation Analysis (CPS disc centrifuge); Integral Sedimentation Analysis (LUMiSizer 12

centrifuge); and Dynamic Light Scattering (Malvern HPPS). In addition, Electron microscopy 13 (SEM and TEM) images of representative nanomaterials have been provided. 14

** average of median values from the three measurement methods. 15 16

According to the applicant, all samples were measured in a standardized fashion according 17 to specific standard operating procedures as follows: 18

19

1. Hydrophilic Powder: 1) Add 30 ml SHMP-solution (0.02 g sodium hexametaphosphate to 20 30 ml deionised water) to 0.2 g titanium dioxide powder in the glass beaker and agitate the 21

sample gently with an overhead or magnetic stirrer for 15 minutes to ensure homogeneity; 22 2) Disperse the probe using an ultrasonic probe (power 50 Watts) for 10 minutes. The 23

ultrasonic horn should not touch the side of the glass beaker or the bottom. The 24 suspensions should be cooled during the dispersion. 25

26 2. Hydrophobic Powder: 1) Add 1 ml isopropyl alcohol to 0.2 g titanium dioxide powder in 27

the glass beaker. To wet the powder slew the beaker carefully; 2) Add 1 drop Disperbyk 28

190 (BYK Chemie, Germany) after adding isopropyl alcohol; 3) Add 30 ml SHMP-solution 29 into the beaker and agitate the sample gently with an overhead or magnetic stirrer for 15 30

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minutes to ensure homogeneity; 4) Disperse the probe using an ultrasonic horn (50 Watts) 1 for 10 minutes. The ultrasonic horn should not touch the side of the glass beaker or the 2

bottom. The suspensions should be cooled during the dispersion. 3 4

3. Oil based Dispersion: Dilute the dispersion to 1% solids by cyclohexane (solids content of 5 dispersion must be supplied by company. 6

7 4. Water based dispersion: Dilute the dispersion to 1% solids by deionised water (solids 8

content of dispersion must be supplied by company). Agitate every sample gently with the 9 stirrer for 1 hour for equilibration before measurement. 10

11

SCCS Comment 12 The different materials included in the dossier have different particle sizes. These range 13

from ~45 nm to 384 nm on volume weighted median basis (average of 3 measurement 14 methods), and ~32 nm to ~102 nm on the basis of number weighted median (average of 3 15

measurement methods). The lower size cut offs (average of 3 measurement methods) 16 range between 18 nm and 63 nm. Note that different methods are typically characterised by 17

systematic, or partially systematic, different measurement uncertainties depending on the 18 size range measured. Therefore the average of different measurement methods on the 19

same nanomaterial does not necessarily provide a more reliable value than measured by an 20

individual method, but has been adopted as a practical approach to size determination. 21 22

23

1.3.11 Microscopy 24

25 An example transmission electron microscopy (TEM) image of TiO2 nanomaterial is shown 26

below: 27 28

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1 2 3

4 An example Cryo-TEM image of TiO2 nanomaterial in formulation is shown below: 5

6

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1 2

3 SCCS Comment 4

The different nanomaterials included in the dossier have primary particles that have either 5 spherical, needle, or lanceolate (longer than wide) shapes, and appear to be present in 6

aggregated clusters. 7 8

1.3.12 Homogeneity and stability 9

10

According to the Applicant, the term “dispersion” has been used in relation to the dispersion 11

of TiO2 clusters/ aggregates in the cosmetic product, whereas aggregates bound by strong 12 forces could not be dissociated. They also claim that coating materials on the TiO2 particle 13

are stable under various conditions of pH, temperature and shear forces, and that the 14 materials used as coating agents for TiO2 may also be present as constitutive ingredients of 15

the same cosmetic product. 16 17

18 SCCS Comments on Physicochemical Characterisation 19

The physicochemical characterisation data provided in the dossier relates to fifteen (15) 20

TiO2 nanomaterials. The data are reasonably extensive, which show that: 21

1. Ten out of the 15 materials (S75-A, S75-B, S75-C, S75-D, S75-E, S75-H, S75-I, S75-22

J, S75-K, S75-L) are rutile. Two other materials (S75-M, S75-N) are mainly rutile with 23 a small proportion (2-5%) of anatase. 24

2. One material (S75-O) is anatase. Two other materials (S75-F and S75-G) are mainly 25 anatase (85%) with rutile (15%). 26

3. The primary crystal size of the materials range between 9 and 21 nm. The average 27 particle sizes in dispersions (measured by 3 different methods) range from ~45 nm to 28

384 nm on volume weighted median basis (average of 3 measurement methods), and 29

~32 nm to ~102 nm on the basis of number weighted median (average of 3 30

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measurement methods). The lower size cut offs (average of 3 measurement methods) 1 range between 18 nm and 63 nm. 2

4. One material (S75-G) is uncoated, all other materials are surface coated with different 3 coating materials (silica, alumina, organo-silanes). 4

5. All coatings are reported to be stable at least in the short-term in vitro test systems. 5 In view of the diversity of the coating materials and some high coating to 6

nanomaterial ratios, it is important to know the concentration of dissolved coating 7 materials, e.g. alumina that could release aluminium ions, in the final formulation. A 8

significant dissolution of a coating material (e.g. alumina) may require a separate 9 safety assessment for the coating material. 10

6. One material (S75-N) is doped with 1000 ppm iron. All other materials are not doped. 11

7. The apparent bulk density of the materials ranges between 0.09 to 0.75 g/cm3. The 12 SCCS notes that the lowest density reported for some materials does not fit in the 13

normal range. As all materials have core particles of TiO2, with sizes in the nano-14 scale, it is not clear why there is such a large variation in their bulk densities. The 15

Applicant needs to clarify whether the materials with low bulk densities have a porous 16 structure, as in such a case they may have different physicochemical properties from 17

the other TiO2 materials. 18

8. One material (S75-E) is in aqueous dispersion. All other materials are either 19

hydrophilic or hydrophobic powders, or are in oil dispersions. 20

9. The VSSAs of the materials range between 192 to 460 m2/cm3 for the different 21 materials, indicating that they are indeed nanomaterials (i.e. VSSA ≥60 m2/cm3). 22

10. Aspect ratios of the different materials range between 1.2 and 4.5, indicating that the 23 high aspect ratio materials have needle or lanceolate shaped particle structures. 24

11. All materials are stated to be photostable. 25

12. UV absorption data for the materials have been provided. 26

13. Zeta potential measurements have been provided for some materials, and not for 27 others due to difficulties in measuring zeta potential for hydrophobic nanomaterials. 28

14. Photocatalytic activity data have been provided for all materials (see Table 2, and 29

corresponding SCCS comments). The data show that the materials have differing 30 levels of photocatalytic activity, which ranges from insignificant to weak (S75-A, S75-31

B, S75-H, S75-I, S75-J, S75-K, S75-L, S75-M, S75-N), to moderate (S75-C, S75-D, 32 S75-E), and strong (S75-F, S75-G; S75-O). All 3 nanomaterials with strong 33

photocatalytic activity are also either anatase form of TiO2, or mainly anatase with 34 some rutile. 35

36

From the physicochemical characterisation data provided, the materials could be broadly 37

grouped as shown below for the purpose of this assessment. This grouping is based on the 38

differences between physicochemical properties and the potential effects of anatase/rutile, 39 coated/uncoated, and photocatalytic/non-photocatalytic forms of TiO2 nanomaterials. It is 40

known that uncoated and non-doped TiO2 nanoparticles are photocatalytic when exposed to 41 UV light. The anatase form has been shown to be more photoreactive than rutile or 42

anatase-rutile mixtures (e.g. Sayes et al., 2006). Another indicator of catalytic activity of 43 nanomaterials is the increased generation of reactive oxygen species (ROS) in biological 44

systems and the resulting toxicological effects, such as cytotoxicity. Jiang et al. (2008) 45 noted that the generation of ROS (per unit surface area) was the highest in amorphous 46

nano-TiO2, followed by anatase, anatase/rutile mixture, and rutile. Anatase form of nano-47

TiO2 has also been reported to be 100 times more cytotoxic under UV than rutile of a 48 similar size (e.g. Sayes et al., 2006). These aspects have already been highlighted in the 49

SCCP opinion on Safety of Nanomaterials in Cosmetic Products (SCCP/1147/07) in the 50 phototoxicity part (page 33): 51

‘When coupled with UV irradiation, anatase TiO2 (hydrophilic, circa 20 nm) was clearly more 52 photogenotoxic than TiO2 (anatase and rutile, both 255 nm) in mouse lymphoma L5178Y 53

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cells, as measured by the comet assay (Nakagawa et al. 1997). Rutile of larger particle size 1 (420 nm) was not photogenotoxic. The nanosized anatase TiO2 was also photogenotoxic in 2

Chinese hamster lung CHL/IU cells, when assessed by chromosome aberration induction, 3 but not in Salmonella typhimurium or in mouse lymphoma L5178Y tk+/- cells, when studied 4

for mutation induction (Nakagawa et al. 1997). Furthermore, this nanosized TiO2 5 (hydrophilic surface) only induced DNA damage, chromosome aberrations and mutations 6

with UV radiation.’ 7

8

9

10 * S75-K and S75L have stated purity of >94% with no impurity profile provided. 11

12

13

On the basis of above physicochemical considerations, the SCCS has considered the TiO2 14

nanomaterials in the following 3 groups of for the purpose of this assessment: 15

- 9 materials (S75-A, S75-B, S75-H, S75-I, S75-J, S75-K, S75-L, S75-M, S75-N) on the 16

basis that they are (mainly) rutile with a relatively low photocatalytic activity. However, 17 two of these materials (S75-K and S75-L) have a stated purity of >94%, with no 18

impurity profile provided. These two materials (S75-K and S75-L) were considered by 19 the SCCS to be not sufficiently pure to include in this opinion. 20

- 3 materials on the basis that they are rutile with a moderate photocatalytic activity 21

(S75-C; S75-D; S75-E); 22

- 3 materials on the basis that they are (mainly) anatase, and also that they have a 23

strong photocatalytic activity (S75-F, S75-G, S75-O). 24

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In view of the foregoing, it is important to note that this opinion applies to all fifteen (15) 1 nanomaterials presented in this submission. The opinion may, however, be also applicable 2

to other TiO2 nanomaterials that have similar characteristics to the 15 nanomaterials in this 3 submission in terms of the physicochemical parameters listed in Tables 1-3, and other 4

specific provisions laid out in Section 2 below. 5

6

1.4 Function and uses 7

8 Titanium dioxide is used as an UV-filter in a concentration up to 25% in cosmetic products. 9

It is regulated in Annex VII, entry 27 of the Cosmetics Directive 10 11

1.5 Toxicological Evaluation 12

13

1.5.1 Acute toxicity 14

15

1.5.1.1 Acute oral toxicity 16

17 Acute toxicity, single oral administration, rat 18

Guideline: OECD Guidelines 401 and EEC Guidelines 92/32/EEC 19

Species/strain: 8 week old rats/Hsd-Win: WU 20 Group size: 5 male/ 5 female 21

Test substance: TiO2 T805, hydrophobic fluffy white powder, CAS 100209-12-9 22 Batch: 27073 23

Purity: TiO2 96.5%, SiO2 3%, carbon approx 4%. 24 Vehicle: suspension in peanut oil 25

Dose levels: 2150 mg/kg 26 Dose volume: 21.5 ml/kg of 100 mg/ml 27

Route: Oral 28

Administration: single dose 29 GLP: yes 30

Study period: August 1993 31 References 32

Submission I - Evonik (Degussa) 1993 (5) and DHS Evonik (Degussa) 1993 (1) 33 34

Results 35 No signs of toxicity recorded during the observation period, no deaths recorded, necroscopy 36

showed no alterations, LD50 for male and female rats >2150 mg/kg. 37

38 SCCS Comment 39

The study relates to S75-F material included in the dossier, which is anatase/rutile material, 40 with organic coating of trimethoxy-caprylylsilane, in oily suspension. This study is relevant 41

to the nanomaterial group (85% anatase, 15% rutile). 42 43

44 Acute toxicity, multiple oral administration, rat 45


Species/strain: 7 week old male rats, 8 week old female rats /Hsd-Cpb: WU 47 Group size: 5 male/ 5 female 48


Purity: TiO2 >97%, Fe2O3 2±1%, carbon 3.5-4.5%. 51

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Vehicle: suspension in olive oil 1 Dose levels: Total dose of 2150 mg/kg (dosed twice in equal amount) 2

Dose volume: twice dose of 21.5 ml/kg of 50 mg/ml 3 Route: Oral 4

Administration: single dose 5 GLP: yes 6

Study period: 7 DHS Evonik (Degussa), 1993 (2) 8

9 Results 10

No signs of toxicity were recorded during the observation period, no deaths recorded, only 11

signs of diarrhoea in 2 male and 1 female rats from day 1 until day 2 after administration. 12 Necroscopy showed no alterations, LD50 for male and female rats were >2150 mg/kg. 13

14 SCCS Comment 15

The study relates to S75-F material included in the dossier, which is a coated, anatase/rutile 16 material, with organic coating of trimethoxy-n-octyl-silane, in oily suspension. This study is 17

relevant to the nanomaterial group (85% anatase, 15% rutile). 18 19

20

Approximate Lethal Dose study, Intragastric intubation, Rats 21 Guideline: OECD Guidelines 401 and EEC Guidelines 92/32/EEC 22

Species/strain: 7 week old Male rats/Crl-CD®BR 23 Group size: not mentioned 24

Test substance: TiO2 T805, white powder, CAS number 13463-67-7 25 Batch: H-20762 26

Purity: TiO2 100%. 27 Vehicle: suspension in deionised water 28

Dose levels: 2,300 to 11,000 mg/kg 29

Dose volume: not described 30 Route: Oral 31

Administration: single dose 32 GLP: No (not mentioned) 33

Study period: August-October 1994 34 Reference: Submission I - DuPont, 1994 (1) 35

36 Results 37

No signs of toxicity were recorded during the observation period, no deaths recorded, 38

pathological examination not performed, weight loss (up to 6%) in some animals after 1 39 day of dosing, ALD >11000 mg/kg, considered as very low toxicity. 40

41 SCCS Comment 42



47

Exploratory study, acute toxicity, oral, mice (Wang et al., 2007) 48 Guideline: OECD Guidelines, No. 420 49

Species/strain: mice/ CD-1 (ICR) 50 Group size: 80 (40 female, 40 male) 51

Test substance: TiO2 nanoparticles (25, 80 and 155 nm) - not mentioned whether 52 rutile or anatase 53

Batch: not mentioned 54 Purity: not mentioned 55

Vehicle: 0.5% hydroxypropylmethylcellulose K4M used as a suspending agent. 56

Dose levels: 5 gram/kg bw 57

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Dose volume: not mentioned 1 Route: single oral gavage 2

Administration: single high dose 5 g/kg bw gavage. 3 GLP: 4

Study period: 5 Reference 213: (Wang, J., Zhou, G., Chen, C., Yu, H., Wang, T., Ma, Y., Jia, G., Gao, Y., Li, 6

B., Sun, J., Li, Y., Jiao, F., Zhao, Y. and Chai, Z. 2007. Acute toxicity and biodistribution of 7 different sized titanium dioxide particles in mice after oral administration. Toxicol Lett 168 8

(2): 176-85). 9 10

Results 11

Retention of a small percentage of titanium (measured by ICP-MS) showed predominantly in 12 the liver and spleen. Kidney, liver and heart pathology was observed with all sizes, with 13

more pronounced effects for 80 and 155 nm particles. Changes in serum biochemical 14 parameters (increased lactate dehydrogenase (LDH) and alphahydroxybutyrate 15

dehydrogenase (alpha-HBDH) levels) were most pronounced for 80 nm particles. 16 17

SCCS Comment 18 The study has a number of flaws, and is therefore of little value to this assessment. 19

Sufficient characterisation of the nanomaterials used was not carried out, the administered 20

dose (5 g/kg/bw) was very high, frequent oesophageal ruptures were reported that led to 21 animal deaths, translocation of TiO2 from GI tract was measured as titanium with no 22

evidence that it was in nanoparticulate form. It is not clear whether any of the effects 23 observed were due to TiO2 toxicity, or simply overloading the gut at high dose of the 24

particulate material. 25 26

27 SCCS Comment on Acute Oral Toxicity 28

The TiO2 nanomaterials tested for this endpoint are mainly anatase/rutile mixtures, coated 29

with trimethoxy-n-octyl-silane. The derived LD50 in rat is >2150 mg/kg. One study has 30 determined the approximate lethal dose at >11000 mg/kg. 31

In addition, the following two articles have been provided on acute toxicity, but they are of 32 no value to this assessment: 33

An article by Ferch, Habersang, 1982 (SI-3) is in fact an old review article (up to 1982) 34 which focuses mainly on the possible health effects of amorphous and crystalline silica. It 35

also includes literature review on possible effects of Degussa P25 TiO2 on the formation and 36 induction of granulomatous changes in the lungs or the peritoneum. Since these were not 37

found, the authors claim that P25 TiO2 is not toxic. As such the article does not provide 38

experimental data, but is solely a review of the literature, with the main emphasis on SiO2 39 and only a few remarks on P25 TiO2. 40

An article by Warheit et al., 2007 (SI-II-215) is a review of different studies on ultrafine 41 TiO2 particles to develop a base set of toxicity tests. As such it does not provide any details 42

on the studies or any experimental data that could be used for this assessment. 43

From the limited data available, the acute oral toxicity of nano-TiO2 (anatase and rutile 44

mixtures) appears to be very low. 45

1.5.1.2 Acute dermal toxicity 46

47

Exploratory study, Acute toxicity and Skin and Eye irritation tests, Mouse and 48 Rabbit 49

50 Guideline: OECD Guidelines 401 and EEC Guidelines 92/32/EEC 51

Species/strain: Male albino mice (acute toxicity tests), and male albino rabbits (skin 52 irritation tests), male albino rabbits (eye irritation tests) 53

Group size: 10 mice for toxicity tests, 4 rabbits for skin irritation tests, 3 rabbits 54

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for eye irritation tests 1 Test substance: TiO2 (referred to as natural colour) 2

Batch: 3 Purity: not stated 4

Vehicle: suspension in water 5 Dose levels: up to 10 g/kg for toxicity study, 100mg/square inch for skin patch 6

tests, 100 mg for eye irritation tests 7 Dose volume: 8

Route: Oral intubation for toxicity tests, skin patch for irritation test, 9 instillation in lower conjunctival sac of eye, 10

Administration: 7 days for toxicity tests, 48 hours for skin irritation tests, eye washed 11

after 5 minutes of instillation. 12 GLP: No 13

Study period: 14 15

Reference 2 16 (Roy, D. and Saha, J. (1981) Acute toxicity of dyes used in drugs and cosmetics, The 17

Eastern Pharmacist, May 1981, pages 125-126) 18 19

Results 20

No mortality recorded in mice, even at 10 g/kg. No sign of skin irritation or eye irritation. 21 LD50 >10,000 mg/kg, TiO2 regarded as non-toxic, non-irritant to both skin and eye. 22

23 SCCS Comment 24

The study is of little value in relation to the current assessment for nano-forms of TiO2 as 25 characterisation data (particle size distribution) have not been provided to show that the 26

tested materials were nanomaterials. 27 28

29

Acute dermal toxicity, limit test, rat 30 Guideline: OECD Guidelines 401 and EEC Guidelines 92/32/EEC 31

Species/strain: 8 week old rats/Sprague Dawley 32 Group size: 5 male/ 5 female 33

Test substance: TiO2 NP88/296 (ultrafine), fluffy white powder, CAS 34 100209-12-9 35

Batch: control No. 27073; July 27th, 93. 36 Purity: TiO2 96.5%, SiO2 3%, carbon approx 4%. 37

Vehicle: suspension in peanut oil 38

Dose levels: 2000 mg/kg 39 Dose volume: 40

Route: Dermal 41 Administration: single application under occlusion 42

GLP: yes 43 Study period: February 1989 44

Submission I 45 Croda (Tioxide UK), 1989 (Reference 6) 46

47

Results 48 No deaths recorded after 24 hour dermal administration, under occlusion, of NP 88/296 at 49

2000 mg/kg. Clinical signs noted only after day 1 of dosing, and included hypokinesia, 50 ataxia, chromodacryorrhoea (eyes and nose), animals hot to the touch. All animals were 51

normal 2 days after dosing. Median Dermal lethal dose (LD50) of NO 88/296 in rats is 52 >2000 mg/kg. No significant abnormalities noted after post-mortem. 53

54 SCCS Comment 55

The study used ultrafine TiO2, and lacks data on characterisation (particle size distribution) 56

of the tested material. According to the Applicant, the material used in this study relates to 57

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rutile material coated with alumina/silica (i.e. S75-A, S75-B, S75-C, S75-L). It is however 1 not clear how the test material relates to those included in the dossier and what proportion 2

of the micronized material was in the nano-scale. 3 4

SCCS Comment on Acute Dermal Toxicity 5 The TiO2 material tested in one study is described as ‘natural colour’. The other study has 6

used ultrafine TiO2, and it is not clear what proportion of the micronized material (coated 7 with alumina/silica) was in the nano-scale. Another reference provided in relation to acute 8

toxicity (Submission I – ref 4, Trochimowicz et al., 1988) is in fact a secondary citation of 9 the oral lethal dose cited in another article which relates to chronic inhalation toxicity. 10

From the provided test data, acute dermal LD50 of TiO2 has been derived at >2000 mg/kg 11

(ultrafine material), and >10,000 mg/kg (natural colour material). However, the provided 12 studies are of no value to the current assessment of nano forms of TiO2. 13

14

1.5.1.3 Acute inhalation toxicity 15

16 No study has been provided on acute inhalation toxicity. The SCCS has therefore considered 17

relevant studies in the open literature: 18 19

Respiratory deposition of particles 20

Inhaled particulate materials may deposit in the lung depending on size (and shape) of the 21 particles, structure of the lung, and breathing pattern (Sarangapani & Wexler, 2000). The 22

mammalian respiratory tract is often divided into three regions - the extrathoracic (mouth 23 or nose and throat), the trachea-bronchial and the alveolar regions with each having a 24

typical structure and function. In general, particles >10 µm deposit in the extrathoracic 25 region. Nanoparticles also mainly deposit in the extrathoracic region, but alveolar deposition 26

has been noted for particles with a size of 300-200 nm down to 3-2 nm (ICRP 1994 – 27 Oberdorster 2005, Cassee et al. 2002). 28

Particulate materials getting into the lung are generally cleared from the respiratory system. 29

Large insoluble particles are cleared mechanically, whereas those that dissolve in the lung 30 are removed via adsorption. Particles in the extrathoracic region are generally removed by 31

coughing or swallowed into the gastrointestinal tract. Particles deposited into the trachea-32 bronchial region are in contact with the mucus layer covering the ciliated cells, and are 33

generally cleared via the ’mucociliary escalator‘, which moves the mucus (and the particles) 34 toward the epiglottis where they are subsequently swallowed and cleared via the GI-tract. 35

Clearing of particles from the alveolar region is much slower and may take weeks to years. 36 The most important pathway here involves alveolar macrophages. These phagocytic cells 37

reside on the alveolar epithelium, and phagocytize the particles. The particle-laden 38

macrophages can be removed via the mucociliary escalator, or can translocate to the 39 interstitial tissue – together with free particles. These clearance mechanisms are similar in 40

humans and most mammals, although clearance rates can significantly differ between 41 species. 42

Some particles may be retained in the alveoli for long periods (months) before being 43 cleared. A small fraction of the inhaled particles can reach the systemic circulation by 44

passing the pulmonary epithelial barrier; another small fraction can probably reach the 45 brain via olfactory nerve route. It has been shown that ultrafine (including nano) particles 46

have a longer retention time in the alveoli compared to larger particles (Oberdorster, 1994). 47

During chronic and/or cumulative exposure nanoparticles in the alveoli potentially 48 accumulate in the tissue of the entire lungs. 49

Exposure to ultrafine particles has been linked to inflammatory and neurodegenerative 50 changes in the olfactory mucosa, olfactory bulb, and cortical and subcortical brain structures 51

(Oberdorster, 2005). So far there are no toxicological studies available which show 52 extrapulmonary effects when the exposure was performed under relevant occupational or 53

environmental conditions. Yet there exists a vast epidemiological literature which clearly 54

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indicates exposures to urban ambient aerosols containing nano-sized particles at high 1 number concentrations are associated with cardiovascular morbidity and mortality (Pope et 2

al., 2009). 3

4

4-Hour Acute Inhalation Toxicity Study in Rats 5 Authors: Dekker, U. 6

Reference: RCC-Report B25007, internal report 7 Guideline: The following guidelines were considered: 8

European Communities, Directive 92/69/EEC, Part B.2 "Acute Toxicity 9 (Inhalation)", published December 29, 1992 and European Communities 10

Directive 93/21/EEC, April 27, 1993 amending the aforementioned 11

Directive. 12 OECD Guidelines for Testing of Chemicals, Section 4, No. 403: "Acute 13

Inhalation Toxicity", adopted May 12, 1981. 14 U.S. Environmental Protection Agency, Health Effects Test Guidelines 15

OPPTS 870.1300, Acute Inhalation Toxicity, August 1998. 16 Species/strain: 15 males and 15 females HanRcc:WIST(SPF) rats; 9-10 weeks old 17

Group size: 15 rats per group, one TiO2 exposed group, one placebo exposed group 18 Test substance: TiO2; 19

Batch: / 20

CAS No. / 21 Purity: unknown 22

Dose levels: A mean TiO2 aerosol concentration of 4.877 mg/L was inhaled by the rats. 23 TiO2 particles were resuspended in water and jet nebulized. Median 24

aerodynamic diameters (MMADs) and geometric standard deviations (GSD) 25 were 1.4 μm (GSD 2.10) 26

Route: Acute 4-hour nose-only inhalation. After a 4-hour inhalation BAL was 27 performed in satellite groups of 5 rats at 14 hours and 2 days after 28

inhalation. The rats were studied at day 15 after exposure. 29

GLP: No 30 Study period: 31

32 Results 33

In BALF collected at 14 hours post end of exposure, total cell count (neutrophil numbers) 34 and total protein were significantly elevated in both sexes of the exposed group compared 35

to the control group. The changes in BALF were consistent with the histopathology findings 36 of diffuse alveolar histiocytosis and alveolar lining cell activation seen in all animals of the 37

exposed group. Significant increases of the absolute and relative lung weights and 38

histopathology findings of diffuse alveolar histiocytosis and alveolar lining cell activation 39 were found in the exposed group on day 2. These findings were consistent with TNFα and 40

IL-6 levels in BALF higher in females of the exposed group than in control group on day 2. 41 42

SCCS Comments 43 It is not clear which of the three noted guidelines were followed. The distribution was not 44

investigated. The deposited TiO2 particle dose was not determined. The exposed group 45 showed signs of inflammation based on the methodology applied. The study was poorly 46

performed and important control parameters are missing. This is by no means a 47

comprehensive study and is of questionable value to this assessment. 48 49

Chronic inhalation Exposure of rats to titanium dioxide dust 50 Authors: Trochimowicz, H.J. et al. (1988) 51

Reference: Chronic inhalation study ref. No. 4 52 Guideline: not specified 53

Species/strain: 3-6 months ChR-CD rats at the begin of the study 54 Group size: 11 males + 11 females 55

Test substance: TiO2 not specified 56

Batch: not specified. 57

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Purity: not specified 1 Dose levels: 250 mg/m³, 50 mg/m³, 10 mg/m³,0 mg/m³, 6h/day, 5 days/week, 104 2

weeks 3 Route: chronic inhalation for 104 weeks; 4

Administration: whole body exposure 5 GLP: not specified 6

Study period: / 7 8

Results 9 After 3 months: alveolar cell hyperplasia at doses of 250 mg/m³, 50 mg/m³, 10

After 6 months: alveolar cell hyperplasia at all dose levels 11

After 12 months: additionally minute areas of collagen fiber deposition at 250 mg/m³ dose 12 After 24 months: massive alveolar hyperplasia, focal patches of pneumonia, areas of 13

collagenized fibrosis; only at 250 mg/m³ dose; occurrence of lung tumours 14 The authors conclude significant patho-physiological alterations at doses of 250 mg/m³, 50 15

mg/m³ but not at 10 mg/m³ 16 17

SCCS Comment 18 This study is one of the early chronic inhalation studies on titanium dioxide which triggered 19

later chronic inhalation studies in the 1980s and 1990s and later investigations into 20

biokinetics and more toxicological endpoints. 21 22

23 Studies in open literature 24

Several sub-chronic (90 days) TiO2 inhalation exposure studies have been reported: 25

- Rats inhaled a TiO2 aerosol of 22 mg/m³ concentration consisting either of 26

nanostructured or pigmentary TiO2 particles for 6h/d 5d/wk for 12 consecutive weeks 27 and were followed up for 1 year (Ferin et al., 1992). 28

- Rats, mice and hamsters inhaled a nanostructured TiO2 aerosol at concentrations of 10, 29

50 or 250 mg/m³ for 6h/d 5d/wk for 13 consecutive weeks and were followed up for 1 30 year (Bermudez et al., 2002; Everitt et al., 2000). 31

- Rats, mice and hamsters inhaled a nanostructured TiO2 aerosol at concentrations of 0.5 32 or 2 or 10 mg/m³ for 6h/d 5d/wk for 13 consecutive weeks and were followed up for 1 33

year (Bermudez et al., 2004) 34

Common findings of these sub-chronic studies were: substantial responses of inflammation 35

and overload associated with diminishing particle clearance in a dose dependent manner, 36 and histologically clear indications of epithelial hypertrophy and hyperplasia. Most 37

pathophysiological responses disappeared after 1 year of recovery and only the very high 38

doses led to persistent adverse effects. Rats always responded more sensitively than mice; 39 hamsters had the least responce. When nanostructured or pigmentary TiO2 particles were 40

compared, stronger effects were observed for the nanostructured particles. 41

Two 5-day inhalation-exposure studies in rats with a follow-up of 28 days as a substitute of 42

sub-chronic 90-days studies with a follow-up of 1 year have been conducted: 43

- TiO2 nanoparticles at a concentration of 100 mg/m³, and pigmentary TiO2 particles at a 44

concentration of 250 mg/m³ - with a positive control exposure to quartz particles at 100 45 mg/m³ (van Ravenzwaay et al., 2009) were investigated. Mild inflammation was 46

reported in lung histology and BAL with subsequent reversibility. All responses were 47

transient but the quartz effects persisted. The authors suggested that the effects seen in 48 these short term studies would be similar to those after 90-day exposure studies. It is 49

however not clear to the SCCS how the major differences seen in these and the other 50 studies can be equated. 51

- Nanostructured TiO2 particles at concentration of 2, 10 and 50 mg/m³ were 52 investigated. Transient inflammatory responses were observed in lung histology and 53

BAL. (Ma-Hock et al., 2009). 54

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Another intratracheal instillation study used nanostructured anatase TiO2 particles of 5, 23 1 and 154 nm (actual hydrodynamic diameters of 19, 28 and 176 nm) at a concentration of 5 2

mg/kg bw administered to the rats and studied until three months after instillation. The 3 results showed that the smaller the particles, the larger the inflammatory response and 4

hypertrophy. However the effects were transient, (Kobayashi et al., 2009). Several other 5 instillation studies have been published that used nano- and submicron-sized TiO2 particles 6

but they have not been considered here because the particles had already formed larger 7 sized agglomerates. 8

9

SCCS Comment on acute inhalation toxicity 10

No study on acute inhalation toxicity was provided. Studies (including open literature) on 11

acute and sub-chronic inhalation exposure to TiO2 nanomaterials have indicated substantial 12 inflammatory responses, and histologically clear indications of epithelial hypertrophy and 13

hyperplasia at high exposure dose. In view of this, the SCCS does not recommend the use 14 of nano TiO2 in applications that would lead to any significant inhalation exposure (e.g. 15

powder or sprayable products). 16 17

1.5.2 Irritation and corrosivity 18

19

1.5.2.1 Skin irritation 20

21 Skin irritation/corrosion, Patch test, Rabbit 22

Guideline: OECD Guidelines 404 and EEC Guidelines 92/32/EEC 23 Species/strain: 11 month old Rabbit/white Russian 24

Group size: 3 male 25 Test substance: TiO2 T805, hydrophobic fluffy white powder, CAS 100209-12-9 26

Batch: 27073. 27 Purity: TiO2 96.5%, SiO2 3%, carbon approx 4%. 28

Vehicle: Paraffin 29

Dose levels: 0.5 g in 0.64 ml paraffin to dorsal skin area patch 6.25 cm2. 30 Dose volume: 31

Route: skin patch 32 Administration: single application, observation over 3 days 33

GLP: yes 34 Study period: August 1993 35

Submission I 36 Evonik (Degussa), 1993 (13) 37

DHS Evonik (Degussa), 1993 (5) 38

Results 39 Very slight erythema (grade 1 in 2 animals), very slight edema (one animal) after one day 40

of exposure. Primary Irritation Index is 0.3, TiO2 was regarded non-irritant on rabbit skin. 41 42

SCCS Comment 43 The study relates to S75-F material included in the dossier, which is anatase/rutile material, 44

with organic coating of trimethoxy-caprylylsilane, in oily suspension. This study is relevant 45 to the nanomaterial group (85% anatase, 15% rutile). 46

47

Skin irritation/corrosion, Patch test, Rabbit 48 Guideline: OECD Guidelines 404 and EEC Guidelines 92/69/EEC 49

Species/strain: 48 month old male, 43 month old female Rabbit/white Russian 50 Group size: 3 (1 male, 2 female) 51

Test substance: TiO2 T817, hydrophobic fluffy white powder, CAS number 52 100209-12-9 53

Batch: 54

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Purity: TiO2 > 97%, Fe2O3 2±1%, carbon approx 3.5-4.5%. 1 Vehicle: 2

Dose levels: 0.5 g in peanut oil to dorsal skin area patch 6.25 cm2. 3 Dose volume: 4


GLP: Yes 7 Study period: February 1998 8

Reference: DHS Evonik (Degussa), 1998 (6) 9 10

Results 11

No changes observed, neither erythema nor edema observed. Primary Irritation Index was 12 0.0, TiO2 regarded non-irritant on rabbit skin. No systemic effects observed. 13

14 SCCS Comment 15

The study relates to S75-F material included in the dossier, which is anatase/rutile material, 16 with organic coating of trimethoxy-n-octyl-silane, in oily suspension. The study is relevant 17


Skin irritation/corrosion, Patch test, Rabbit 20

Guideline: OECD Guidelines 404 and EEC Guidelines 92/32/EEC 21 Species/strain: 11 month old Rabbit/ New Zealand white 22

Group size: 3 male, 3 female 23 Test substance: TiO2 H20762, CAS number 13463-67-7 24

Batch: 25 Purity: TiO2 100%. 26

Vehicle: 27 Dose levels: 0.5 g in pre-moistened patch (2 inch square gauze) 28

Dose volume: 29


GLP: No (not mentioned) 32 Study period: August-September 1994 33

Reference: Submission I - DuPont, 1994 (10) 34 35

36 Results 37

Three rabbits showed no dermal irritation during the study, no to mild erythema by 1 hour 38

after patch removal. By 24, 48 and 72 hours, no to slight erythema observed, no edema 39 observed during the study. H-20762 is regarded a mild skin irritant. 40

41 SCCS Comment 42

The study is of little value in relation to assessment for nano-form of TiO2 as there is a lack 43 of data on characterisation (particle size distribution) of the tested materials to show that 44

they were nanomaterials. 45 46

Skin irritation/corrosion, Patch test, Rabbit 47

Guideline: not mentioned 48 Species/strain: Rabbit/ albino 49

Group size: 6 male 50 Test substance: TiO2 - referred to as Haskell Nos. (H 12684, H 12685, H 12686) 51

Batch: 52 Purity: not mentioned 53

Vehicle: 54 Dose levels: 0.5 g pre-moistened with physiological saline (1½ inch square 55

gauze) 56

Dose volume: 57

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GLP: No (not mentioned) 3 Study period: 4

Reference: Submission I DuPont, 1978 (11) 5 6

Results 7 No skin irritation observed on intact rabbit skin. 8

9 SCCS Comment 10

The study is of little value in relation to assessment for nano-form of TiO2 as there is a lack 11

of data on characterisation (particle size distribution) of the tested materials to show that 12 they were nanomaterials. 13

14 Skin irritation/corrosion, Patch test, guinea pig 15

Guideline: not mentioned 16 Species/strain: guinea pig/ albino 17

Group size: 12 male 18 Test substance: TiO2 - referred to as 99.5% active ingredient 19

Batch: 20

Purity: 21 Vehicle: 22

Dose levels: 0.5 g powder and 0.1 g 28% paste were slightly rubbed into 23 shaved back skin, covered with impervious film and wrapped. 24

Dose volume: 25 Route: skin patch 26

Administration: single application, 24 hours, then rinsed in water, observation 27 over 2 days 28

GLP: No (not mentioned) 29

Study period: 30 Reference: Submission I - DuPont, 1969 (12) 31

32

Results 33

No skin irritation observed on intact guinea pig skin. 34

35

SCCS Comment 36 The study is of little value in relation to assessment for nano-form of TiO2 as there is a lack 37

of data on characterisation (particle size distribution) of the tested materials to show that 38

they were nanomaterials. 39

40

Skin irritation/corrosion, 5 day repeat application study, Rabbit 41 Guideline: not mentioned 42

Species/strain: Rabbit 43 Group size: 2 male, 1 female 44

Test substance: TiO2 utrafine dispersion - referred to as NP 89/97, NP 89/98. 45 Batch: 46

Purity: not mentioned 47

Vehicle: 48 Dose levels: around 0.5 g (2.5 cm2 patch) 49

Dose volume: around 0.5 ml 50 Route: skin patch 51

Administration: 4x repeated (application, removal, skin observation) 52 GLP: No 53

Study period: 54 Reference: Submission I - Croda (Tioxide UK), 1989 (14) 55

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1

Results 2

One animal died on day 4 (unrelated to the test), 5 day repeat applications produced mean 3 irritation scores of 1.58 and 1.92 for 89/97, NP 89/98 respectively. NP 89/98 considered 4

slightly more irritant than NP 89/97. 5 6

SCCS Comment 7 The study used ultrafine TiO2, however, data on characterisation (particle size distribution) 8

of the tested material has not been reported. It is therefore not clear whether the material 9 had a nano-sized fraction, and if so, in what proportion. 10

11

Skin irritation/corrosion, 5 day repeat application study, Rabbit 12 Guideline: not mentioned 13

Species/strain: Rabbit/ New Zealand white 14 Group size: 3 (2 male, 1 female) 15

Test substance: TiO2 utrafine dispersion - referred to as NP 88/296. 16 Batch: 17

Purity: not mentioned 18 Vehicle: 19

Dose levels: 2 dispersions tested (40% A.I. and 10% A.I. which was diluted 20

with carrier oil NP88/310) 21 Dose volume: around 0.5 ml 22

Route: skin patch 23

Administration: 4x repeated (application, removal, skin observation) 24

GLP: No (not mentioned) 25 Study period: 26

Reference: Submission I - Croda (Tioxide UK), 1989 (15) 27

28

Results 29 5 day repeat applications produced mean irritation scores of 0.13 for both dispersions 30

tested (i.e. no dose response). Neither the undiluted or diluted test material NP 88/296 31 produced significant reactions. One rabbit did not react, and the other 2 rabbits showed only 32

slight to non persistent erythema. 33

34 SCCS Comment 35

The study used ultrafine TiO2. However, there is a lack of data on characterisation (particle 36 size distribution) of the tested material. According to Applicant, the material used in this 37

study relates to rutile material coated with alumina/silica (i.e. S75-A, S75-B, S75-C, S75-L). 38 However it is not clear how the test material relates to the nanomaterials included in the 39

dossier and what proportion of the micronized material was in the nano-scale. 40 41

SCCS Comment on Skin irritation 42

The study by Warheit et al., 2007 (SI-II-215) is of no use to this assessment because it is a 43 detailed literature review on the possible effects of different TiO2 ultrafine particles. As such 44

it does not provide details on the studies, or any experimental data, that could be used for 45 this assessment. 46

Two studies provided in the submission are relevant to the TiO2 nanomaterials. They relate 47 to anatase/rutile mixture, coated with trimethoxy-n-octyl-silane. In one of the studies, the 48

test animals showed signs of very slight erythema and oedema. The primary irritation index 49 was estimated to be zero and 0.3, and the materials regarded as non-irritant on rabbit skin. 50

Two other studies used ultrafine grade materials and showed the mean irritation scores of 51

0.3 and 1.58-1.92 during 5 day repeat applications on rabbit skin, but the proportion of 52 nano-scale fraction in the materials used has not been reported. 53

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The remaining 3 studies showing the tested materials as either mild irritant or non irritant 1 to rabbit and guinea pig skin are of little value to this assessment because there is a lack of 2

data on characterisation (particle size distribution) of the tested materials, and it is not clear 3 whether they were in fact nanomaterials. 4

From the limited useful data presented in the dossier, it appears that the TiO2 5 nanomaterials are either mild or non-irritant to skin. 6

7

1.5.2.2 Mucous membrane irritation 8

9 Eye irritation, single application, rabbit 10

Guideline: OECD Guidelines 405 (1) and EEC Guidelines 92/32/EEC 11

Species/strain: 10-11 month old Rabbits/ white Russian (albino) 12 Group size: 3 (males) 13


Purity: TiO2 96.5%, SiO2 3%, carbon approx 4%. 16 Vehicle: 17

Dose levels: 22.8 to 24.3 mg 18 Dose volume: 0.1 ml 19

Route: eye instillation 20

Administration: single application, 3 days observation period 21 GLP: Yes 22

Study period: August 1993 23 Reference: Submission I - Evonik (Degussa), 1993 (9); DHS Evonik 24

(Degussa), 1993 (3) 25 26

Results 27 No alterations detected in cornea, iris and conjunctiva, primary irritation index is zero, TiO2 28

(805) regarded as non-irritant on rabbit eye. No systemic toxic effects detected. 29

30 SCCS Comment 31



Eye irritation, single application, rabbit 36 Guideline: OECD Guidelines 405 (1) and EEC Guidelines 92/69/EEC 37

Species/strain: 35 month old Rabbits/ white Russian (albino) 38

Group size: 3 (females) 39 Test substance: TiO2 T817, hydrophobic fluffy white powder, CAS number 40

100209-12-9 41 Batch: 04095 42

Purity: TiO2 >97%, Fe2O3 2±1%, carbon 3.5-4.5%. 43 Vehicle: 44

Dose levels: 11.5 to 16.8 mg 45 Dose volume: 0.1 ml 46

Route: eye instillation 47

Administration: single application, 3 days observation period 48 GLP: Yes 49

Study period: February 1998 50 Reference: DHS Evonik (Degussa), 1993 (4) 51

52 Results 53

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Some blood vessels definitely hyperaemic in two animals after one hours of application. 1 Primary irritation index is 0.3, TiO2 regarded as non-irritant on rabbit eye. No systemic 2

toxic effects detected. 3 4


coated with organic coating of trimethoxy-n-octyl-silane, in oily suspension. This study is 7 relevant to the nanomaterial group (85% anatase, 15% rutile). 8

9 10

Eye irritation, single application, rabbit 11

Guideline: OECD Guidelines 405 (1) and EEC Guidelines 92/69/EEC 12 Species/strain: Rabbits/ New Zealand white 13

Group size: 2 (females) 14 Test substance: TiO2 H-20762, CAS number 13463-67-7 15

Batch: 16 Purity: TiO2 100%. 17

Vehicle: 18 Dose levels: approx. 10 mg 19

Dose volume: 20

Route: eye instillation 21 Administration: single application, eye washed after 20 seconds of application. 3 22

days observation period 23 GLP: yes 24

Study period: September 1994 25 Reference: Submission I - DuPont, 1994 (7) 26

27 Results 28

Moderate redness and slight chemosis observed in both treated and untreated washed eyes 29

(normal after 1 and 3 days respectively). No clinical signs of toxicity obeserved, TiO2 30 (H20762) regarded as moderate eye irritant but could be classified as non-irritant under the 31

EEC Directive 93/21, Annex VI. 32 33

SCCS Comment 34 The study is of little value in relation to assessment for nano-form of TiO2 as there is a lack 35

of data on characterisation (particle size distribution) of the tested materials to show that 36 they were nanomaterials. 37

38

39 Eye irritation, single application, rabbit 40

Guideline: OECD Guidelines 405 (1) and EEC Guidelines 92/69/EEC 41 Species/strain: Rabbits/ New Zealand white 42

Group size: 3 (2 male, 1 female) 43 Test substance: TiO2 NP 88/296 (ultrafine) 44

Batch: not mentioned 45 Purity: not mentioned 46

Vehicle: 47

Dose levels: not mentioned 48 Dose volume: 0.1 ml 49

Route: eye instillation 50 Administration: single application, eye washed after 20 seconds of application. 3 51

days observation period 52 GLP: Yes 53

Study period: 54 Reference: Submission I - Croda (Tioxide UK), 1989 (8) 55

56

Results 57

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No corneal or iridial reactions, slight conjunctival redness (score 1) which disappeared after 1 72 hours of treatment. TiO2 (NP88/296) is regarded slightly irritant to rabbbit eyes. 2

3 SCCS Comment 4

The study relates to ultrafine TiO2. However, information on the characterisation (particle 5 size distribution) of the tested material has not been reported. According to the Applicant, 6

the material used in this study relates to rutile material coated with alumina/silica (i.e. S75-7 A, S75-B, S75-C, S75-L). It is however not clear how the test material relates to the 8

nanomaterials included in the dossier and what proportion of the micronized material was in 9 the nano-scale. 10

11

SCCS Comments on Eye Irritation 12 The following two articles provided with the submission on acute toxicity are of no value to 13

this assessment: 14 1. An article by Frosch and Kligman (Reference 16 - S75 irritation skin) refers mainly to 15

the development of a scarification chamber test for irritancy of materials. It does 16 refer irritancy of titanium dioxide as low, but it is not clear whether the tested TiO2 17

was a nanomaterial. 18

2. An article by Warheit et al., 2007 (SI-II-215) is a review of different studies on 19

ultrafine TiO2 particles to develop a base set of toxicity tests. As such it does not 20

provide any details on the studies or any experimental data that could be used for 21 this assessment. 22

Two other studies provided used TiO2 anatase/rutile mixtures, coated with trimethoxy-n-23 octyl-silane. From these studies, primary irritation index was between zero and 0.3. Another 24

study has regarded the tested material (TiO2-NP88/296) as slightly irritant to rabbit eye. In 25 this study, the material used has been described as ultrafine rutile material coated with 26

alumina/silica (relating to S75-A, S75-B, S75-C, S75-L) but information on characterisation 27 (particle size distribution) has not been reported to indicate what proportion was in the 28

nano-scale. Similarly, another study has regarded the tested material (TiO2-H20762) 29

moderately irritant to rabbbit eye, but it is not clear whether the tested material was a 30 nanomaterial. 31

From the limited useful data provided, eye irritation potential of nano-TiO2 appears to be 32 low. 33

34

1.5.3 Skin sensitisation 35

36 Skin sensitisation, Guinea Pig, maximisation test 37


Species/strain: 8 week old 12 male, 10 female guinea pigs/Pirbright white 39 Group size: 3 (1 male, 2 female) 40


Purity: TiO2 96.5%, SiO2 3%, carbon approx 4%. 43 Vehicle: paraffin oil, Freunds Complete Adjuvant for immunisation 44

Dose levels: 0.5 g in paraffin oil to dorsal skin area 5 cm2 patch. 45 Dose volume: 0.1 ml of 0.5% dispersion, 0.2 ml of 5% dispersion for 46

challenge 47

Route: Induction application intradermal and epidermal, challenge 48 application epidermal 49

Administration: single application, 48 hours, challenge on day 22 for 24 hours, 50 observation over 48 hours 51

GLP: yes 52 Study period: June 1992 53

Reference: Submission I Evonik (Degussa), 1992 (19); DHS Evonik 54 (Degussa), 1992 (7) 55

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1

2

Results 3 Following epidermal challenge neither treated nor control animals showed any changes at 4

the skin. TiO2 regarded as non-sensitiser in maximisation test on guinea pig skin. No 5

systemic effects observed. 6 7

8 SCCS Comment 9



Skin sensitisation, Guinea Pig, Buehler test 14

Guideline: OECD Guidelines 406 and EC Guidelines 96/54/EC 15 Species/strain: 8 week old guinea pigs/PsdPCC: DH 16

Group size: 20 male, 20 female (2 vehicle control groups of 10, and 1 test 17 group of 20) 18


Purity: TiO2 > 97%, Fe2O3 2±1%, carbon approx 3.5-4.5%. 21 Vehicle: paraffin oil 22

Dose levels: 0.5 g applied, 3 applications on day 1,8,15. 23

Dose volume: 24 Route: Induction phase duration 15 days, epidermal, challenge 25

application epidermal (occlusive patch) 26 Administration: epidermal, 48 hours, challenge on day 29 for 6 hours, 27

observation over 48 hours. 28 GLP: yes 29

Study period: November-December 1997 30 Reference: DHS Evonik (Degussa), 1992 (8) 31

32

Results 33 Following first challenge, 3 out of 10 animals reacted with an erthyema and 1 in 10 animals 34

showed edema. Following epidermal challenge neither treated nor control animals showed 35 any changes at the skin. TiO2 regarded as non-sensitiser in Buehler test on guinea pig skin. 36

No systemic effects observed. 37 38


coated with organic coating of trimethoxy-n-octyl-silane, in oily suspension. This study is 41

relevant to the nanomaterial group (85% anatase, 15% rutile). Due to the absence of skin 42 penetration of TiO2 as demonstrated by many studies included in this dossier, the 43

usefulness of the Buehler test for assessing sensitisation potency of nanomaterials is 44 doubtful as it is based on exposure to intact skin. 45

46 Skin sensitisation, Guinea Pig, Magnusson-Kligman maximisation test 47

Guideline: 48 Species/strain: guinea pigs/Dunkin Hatley strain 49

Group size: 20 test group, 16 control group 50

Test substance: TiO2 NP89/145 51 Batch: 52

Purity: TiO2 96.5%, SiO2 3%, carbon approx 4%. 53 Vehicle: Freunds Complete Adjuvant for immunisation 54

Dose levels: 2 cm x 4 cm patch, 2cm x 2 cm patch for challenge 55 Dose volume: 56

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Route: Induction with NP 89/145 at 10% v/v in NP 88/310 (injection) 1 and 100% (topical), challenge application at 100% and 50% v/v 2

in NP88/310. 3 Administration: Patch, 48 hours (induction patch), 24 hour (challenge patch), 4

observation period 24 and 48 hours 5 GLP: Yes 6

Study period: April-May 1989 7 Reference: Submission I - Croda (Tioxide, UK), 1989 (20) 8

9 Results 10

At challenge, none of the test or control group animals treated with NP 89/145 at 100% or 11

50% v/v (in NP 88/310) showed a positive response. No evidence that NP 89/145 is a 12 sensitiser in guinea pigs. Classified as a weak sensitiser according to the Magnusson-13

Kligman classification. No clinical signs were noted, body weight gains were acceptable. 14 15

SCCS Comment 16 The study used ultrafine TiO2, however, there is a lack of information on the 17

characterisation (particle size distribution) of the tested material. According to Applicant, 18 the material used in this study relates to rutile material coated with alumina/silica (i.e. S75-19

A, S75-B, S75-C, S75-L). It is however not clear how the test material relates to the 20

nanomaterials included in the dossier because the proportion of the nano fraction in the 21 micronized material has not been provided. 22

23 SCCS Comment on Skin Sensitisation 24

The article by Warheit et al., 2007 (SI-II-215) is a review of different studies on ultrafine 25 TiO2 particles to develop a base set of toxicity tests. As such it does not provide any details 26

on the studies or any experimental data that could be used for this assessment. 27

From two of the other studies, TiO2 nanomaterials (anatase/ rutile mixture, coated with 28

trimethoxy-caprylylsilane or trimethoxy-n-octyl-silane) have been regarded non-sensitiser. 29

Another material (rutile, coated with alumina/silica) is classified as a weak sensitiser 30 according to the Magnusson-Kligman classification (that considers 0 to 8% response a weak 31

sensitizer category). The material used in this study is described as ultrafine rutile material 32 coated with alumina/silica (relating to S75-A, S75-B, S75-C, S75-L) but information on 33

characterisation (particle size distribution) of the tested materials has not been reported to 34 indicate what proportion was in the nano-scale. 35

Due to the absence of skin penetration of TiO2 as demonstrated by many studies included 36 in this dossier, the usefulness of the Buehler test for assessing sensitisation potency of 37

nanomaterials is doubtful as it is based on exposure to intact skin. 38

From the limited useful data, TiO2 nanomaterials appear to be weak or non- skin sensitiser. 39

40

1.5.4 Dermal / percutaneous absorption 41

42

In vitro studies: 43 44

Guideline/method: 45 Species: human abdominal epidermis 46

Test substances: Titanium dioxide T805, comprising 5% micronized titanium 47

dioxide; not radiolabelled. 48 Particle size: not given 49

Group sizes: 2 female donors in experiment 1, 1 male and 1 female donor in 50 experiment 2 51

Dose applied: 3.6g/cm2 of cream with a content of 5% micronized titanium 52 dioxide (actual dose 3.55 mg/cm2) 53

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Skin area: 0.32 cm2 1 Skin temperature: 30-32°C 2

Test chamber: flow through diffusion cells 3 Receptor fluid: 0.9% saline 4

Exposure period: 6 hours 5 GLP: yes 6

Published: no 7 Study period: 1995 8

Reference: Reference 24 submission 1 9 10

Method 11

The amount applied to each cell was 3.55 mg/cm2. Skin integrity was checked. The 12 penetration through the skin membranes was determined over a period of 6 hours under 13

non-occluded conditions. The receptor fluid was delivered at a flow rate of about 1.5 mL/h 14 during the testing period. The perfusate from each cell was collected separately at ambient 15

temperature for 0-8h post application. Eight hours post application the perfusate sampling 16 was terminated. All skin membrane rinse fractions were combined according to the 17

individual cells and added to the 0-8h perfusate. 18 19

Results 20

The perfusate samples were analysed by IPCMS, the TiO2 content ranged from 2.6 to 4.8 21 ng/ml. These concentrations were reported to be in the same range as the ‘blind’ solutions 22

(2.-2.9 ng/ml). Transmission electronic microscopy of titanium dioxide in the skin samples 23 showed presence only in the outer skin layers and not in the deeper layers of the epidermis. 24

Thus TiO2 nanoparticles did not penetrate through human skin under the experimental 25 conditions described above. 26

27 SCCS Comments 28

The study shows lack of detectable skin penetration of the test nanomaterial which relates 29

to S75-F included in the dossier (anatase/rutile material, with organic coating of 30 trimethoxy-caprylylsilane, in oily suspension). This study is relevant to the nanomaterial 31

group (85% anatase, 15% rutile). 32 The particle size of the tested nano-material was not determined in this study. It is assumed 33

that the particle size is similar to the data shown in Table 1.3. However most likely the 34 particles were present as agglomerates as the test item was used in a cream formulation. 35

36 Study Design: 37

Guideline/method: - 38

Species: human abdominal epidermis 39 Test substances: micronized TiO2: Eusolex TA (5% O/W lotion), 40

micronized TiO2: Eusolex TC (5% W/O cream) 41 vehicle (O/W lotion and W/O cream) 42

Particle size: particle sizes not provided, Eusolex TA: BET= 84.2 m2/g Eusolex TC: 43 BET= 58.8 m2/g 44

Group sizes: 4 cells per donor; 4 donors 45 Dose applied: between 3.19 and 4.28 mg/cm2 46

Skin area: 0.32 cm2 47

Skin temperature: 30-32°C 48 Test chamber: flow through diffusion cells 49

Receptor fluid: 0.9% saline 50 Exposure period: 6 hours 51

GLP: yes 52 Published: no 53

Study period: 1995 54 Reference: Reference 25 submission 1 55

56

Method 57

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35

The amount applied to each cell was 3.19-3.31 mg/cm2 (Eusolex TC and TA, respectively; 1 applied amount of vehicle only was slightly higher). Skin integrity was checked. The 2

penetration through the skin membranes was determined over a period of 6 hours under 3 non-occluded conditions. The receptor fluid was delivered at a flow rate of about 1.5 mL/h 4

during the testing period. The perfusate from each cell was collected separately at ambient 5 temperature for 0-8h post application. 6

Eight hours post application the perfusate sampling was terminated. All skin membrane 7 rinse fractions were combined according to the individual cells and added to the 0-8h 8

perfusate. 9 10

Results 11

The perfusate samples were analysed by ICP-OES, the TiO2 content were below 12 0.05ug/sample. No, or only slight traces of TiO2 particles were detectable on the skin 13

samples treated with Eusolex® TA under the light microscope. The refracting colourless 14 TiO2 particles were localized on the outer surface of the stratum corneum. One skin sample 15

revealed two particles sited intracellularly at one location at the stratum granulosum. 16 Whether these were refracting particles of TiO2 could not be resolved unequivocally under 17

the optical microscope. Multiple foci of TiO2 particles were observed on most of the skin 18 samples that had been treated with Eusolex® TC. The refracting particles were localized on 19

the outer surface of the stratum corneum. It was concluded that titanium dioxide 20

nanoparticles did not penetrate through human skin under the experimental conditions 21 described above. 22

23 SCCS Comments 24

The study shows lack of detectable dermal penetration of TiO2 nanoparticles. The test 25 material possibly (as it is not clear from the different code) relates to S75-M, S75-N, and/or 26

S75-O. The particle size of the tested nano-material was not determined in this study. 27 28

29

Test for penetration of micronized TiO2 through the egg membrane or the chorio-30 allantoic membrane (CAM). 31

Guideline: 32 Species/strain: White Leghorn chicken eggs, freshly fertilized 33

Group size: 3 eggs per group (control group: 2 eggs) 34 Test substance: micronized Eusolex TC (TC); 35

Batch: TO 118279 36 Purity: not reported 37

Particle size: not reported 38

GLP: 39 Reference: Reference 26 submission I 40

41 Method 42

The testing material was prepared on the day of exposure. The concentration was 5 g/100 43 ml carrier. The carrier used was water for injection to which 0.01 % of the cationic tenside 44

UCARE 10 had been added. To enable the test material to be applied to the egg membrane, 45 the eggshell was opened with the aid of a dentist's drill and the material was introduced 46

with the aid of a needle. The volume introduced was 0.06 ml per egg. To enable the 47

material to be applied to the CAM (chorio-allantoic membrane), the eggshell was taken off, 48 the egg membrane removed and the material introduced onto the exposed CAM. The 49

volume introduced was 0.3 ml. After the prescribed period of exposure, the treated surface 50 was fixed for 24h with approximately 10% formaldehyde solution. The fixed CAM or egg 51

membrane with CAM was removed, embedded in paraffin, sliced, and then stained with 52 nuclear fast red and H. E. The sections were evaluated under an optical microscope. 53

54 Results 55

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36

No signs of penetration by TiO2 through the egg membrane or the chorio-allantoic 1 membrane were seen under an optical microscope. The introduction of TiO2 was fully 2

tolerated in this sensitive model. 3 4

SCCS Comments 5 The test report is very concise. No positive control was used in this test. This test is 6

therefore of very limited use for this assessment. 7 8

9 Study Design: 10

Guideline/method: 11

Species: human abdominal skin 12 Test substance: J&J Baby Sunblock SPF 30 (2723L) containing microfine titanium 13

oxide (Hombifine 535) (conc unknown) 14 Particle size: not reported. 15

Group sizes: not reported (1 donor?) 16 Dose applied: 400 um formulation 17

Skin area: not reported 18 Skin temperature: not reported 19

Test chamber: flow through diffusion cells 20

Receptor fluid: 0.9% saline 21 Exposure period: 24h hours 22

GLP: no 23 Published: no 24


27 Method 28

A layer of about 400 um of formulation was applied on each human cadaver skin sample 29

and left to dry for 15 minutes. The treated skin samples with the epidermis side facing up 30 were then mounted on each of the modified diffusion cells. The receptor compartment was 31

filled with 0.9% NaCl adjusted to pH 7.4 and 5 respectively. The permeation was conducted 32 for 24 hrs and the receptor solutions were collected at the end of the experiment. The 33

amount of cream left on the skin surface was then recovered using wipes and rinsed with 34 methanol (methanol washings). 35

36 Results 37

In these diffusion cell based tests, samples of stripped human cadaver skin and mouse skin 38

were used. The stripped skin does not have a stratum corneum and can thus be regarded to 39 simulate injured skin. The study showed that only a negligible amount of titanium 40

permeated through either whole skin or the simulated "damaged skin". About 15% of 41 titanium oxide was found in the skin tissue and most of the titanium (ca. 85%) was 42

recovered from the skin surface for both whole skin and stripped skin when the receptor pH 43 was adjusted at pH 7. 4. It appears that titanium has little tendency to permeate through 44

the skin. The amount of titanium oxide recovered in the skin tissue may include the physical 45 adsorption of titanium oxide to the skin surface, which was difficult to be rinsed off by 46

methanol. 47

The effect of pH in the receptor fluid may play an important role towards the penetration of 48 titanium oxide. The point of zero charge (pzc) of microfine titanium oxide (Hombifine S35) 49

is 5.6. Therefore, the receptor fluid will provide better "sink" conditions if its pH is adjusted 50 further away from 5. 6. Less titanium was found in the skin when the receptor pH was 51

controlled at pH 5. This can be explained by the fact that pH 5 (0.6 pH unit away from 52 pzc) is providing less "sink condition" compared to pH 7.4 (1.8 pH unit from zpc). It was 53

concluded that the chance for titanium oxide to penetrate across human cadaver skin is 54 slim. 55

56

SCCS Comments 57

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37

This is a special and limited study to investigate the influence of different pH conditions. 1 Reporting is very concise. Therefore this study provides some additional but limited 2

information for the risk assessment. 3 4

5 Guideline/method: 6

Species: human abdominal skin 7 Test substances: Sunscreen cream with 5% UV-Titan M160 formulation containing 5% 8

titanium dioxide Sunscreen cream without UV-Titan (ca 50 ml). 9 Particle size: not given 10

Group sizes: 3 donors, 1 male and 2 females; 17 samples from 3 donors were 11

treated with sunscreen cream with UV -titan M 160 formulation. A 12 total of 4 samples of epidermis taken from the 3 different donors 13

were treated with the control formulation 14 Dose applied: 2.06 mg/cm2 of cream with a content of 5% micronized titanium 15

dioxide 16 Skin area: 0.32 cm2 17

Skin temperature: 30-32°C 18 Test chamber: flow through diffusion cells 19

Receptor fluid: 0.9% saline 20

Exposure period: 8 hours 21 GLP: yes 22

Published: no 23 Study period: 1996 24

Reference: Reference 30 submission 1 25 26

Method 27 The amount applied to each cell was 2.06 mg/cm2. Skin integrity was checked. The 28

penetration through the skin membranes was determined over a period of 6 hours under 29

non-occluded conditions. The receptor fluid was delivered at a flow rate of about 1.5 mL/h 30 during the testing period. The perfusate from each cell was collected separately at an 31

ambient temperature for 0-8h post application. Eight hours post application the perfusate 32 sampling was terminated. 33

34 Results 35

The absorbed amount of Titanium Dioxide was below the detection limit of 5 ng (1ug/l in 36 ICP-MS) in all samples. The analyses of the samples did not indicate significant penetration 37

of Titanium Dioxide UV-TITAN within the detection limit of the method. 38

39 SCCS Comments 40

The study shows lack of detectable dermal penetration of TiO2 nanoparticles. The tested 41 material is S75-I (>99.5% Rutile, coated with 7% alumina 10% stearic acid). 42

43 44

Guideline/method: 45 Species: Human (4 females, mean age 26), upper arm 46

Test substances: A- Oil/Water lotion: 5% w/w Ti02 from 12.5% Tioveil AQG 47

B- Water/Oil cream: 7.5% w/w Ti02 from 18.75% Tioveil TG 48 C- Oil/Water lotion: 7.5% w/w Ti02 from 18.75% Tioveil OP 49

Particle size: not reported 50 Group sizes: 4 volunteers, 3 different locations of the upper arm (for application A, 51

B and C) 52 Dose applied: 2.0 ul/cm2 : 8 ul spread over 4 cm2 area of skin. 53

Skin: Intact human skin 54 Skin temperature: 37 °C 55

Exposure period: 8h under occlusion 56

GLP: No 57

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3 Method 4

The three test products were randomly allocated to three of the four test sites on the 5 forearm. After the 8 hour occlusion, the dressings were removed. The sites were not wiped 6

prior to removal of stratum corneum by the skin surface biopsy (SSB) procedure. 7 Successive SSBs were taken from the same site such that a profile across the stratum 8

corneum was obtained. Four SSBs were taken from each of the treated sites. 9 The migration of titanium dioxide from sunscreen formulas into the skin was investigated 10

using a range of sunscreen formulas (A-C) and four subjects. Consecutive 4cm2 skin 11

samples (biopsies) were taken from test areas. A maximum of 4 biopsies were taken from 12 any one skin area, providing 16-20 skin layers in total. Selected skin biopsies were then 13

analysed using X-ray microanalysis to determine the concentration of titanium dioxide in the 14 biopsy and to show the migration of the titanium dioxide through the skin. 15

16 Results 17

Emulsion A did not appear to have migrated past the first biopsies from subjects 1, 2 and 4 18 but had migrated to the second biopsy from subject 3. 19

Comparing the emulsions tested on subject 1, emulsions A and B showed little difference 20

with titanium only present in the first biopsy, but the titanium from emulsion C had 21 migrated to the second biopsy. These results have been confirmed by transmission electron 22

microscopy examination of these samples where titanium dioxide crystals were shown to be 23 present through the first biopsy and in the second skin biopsy of the area treated with 24

emulsion C but not in the second biopsies of the areas treated with any of the other 25 emulsions. Repeat analyses on selected samples showed that there was an error of ±0.2% 26

in the measurement of titanium in these samples. This indicates that there is some variation 27 across the samples possibly due to uneven migration of the sunscreen or uneven thickness 28

of the biopsies. The detection limit of the analyser was -0.1% and though comparative 29

results were obtained by this method it is not as accurate as observations made by 30 transmission electron microscopy. Any measurements less than 0.3% were confirmed by 31

repeat analyses. It was concluded that in all cases no Ti02 was detected beyond the top two 32 (out of four) skin surface biopsies. No evidence of penetration to the viable epidermis was 33

found. 34 35

SCCS Comments 36 The study shows some penetration of TiO2 nanoparticles to the outer layers of skin, but not 37

to the viable epidermis. The tested material relates to S75-B (>99.5% Rutile, coated with 38

6% silica, 16% alumina). 39 40

41 Study Design: 42

Guideline/method: Comparative study according to an internal laboratory methodology 43 considering real use conditions and recommendation of US FDA and 44

COLIPA SPF requirements 45 Species: Human (25- to 65-year-old adults) 46

Test substances: Commercial products containing coated (Al2O3 and SiO2) nano-sized 47

titanium dioxide. No information on size except for Eusolex T-2000 48 TiA: contained only TiO2 49

TiB: contained TiO2 plus ZnO 50 TiHB: (Eusolex T-2000) contained coated rutile TiO2 (average size of 51

20 nm) 52 Particle size: Nanoparticles of TiO2 needle-shaped; dimension not given 53

Group sizes: TiA, TiHB: 8 volunteers (intact skin) 54 TiB: 9 volunteers (intact skin) 55

TiA, TiB, TiHB: 10 volunteers (stripped skin) 56

TiA: 4 psoriatic patients 57

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39

Controls: 6 volunteers for basal elemental concentration in the skin 1 Dose applied: 0.5 – 1.0 mg/cm² on an area of 25 cm² 2

Skin: Intact and tape stripped human skin 3 Skin temperature: 37 °C 4

Exposure period: 2 h (intact) or 48 h (stripped skin and psoriatic patients) 5 GLP: No 6

Published: Yes 7 Study period: Before 2009 8

Reference: Filipe et al., 2009 (54, 155) 9 10

Method 11

The localization and possible skin penetration of TiO2 nanoparticles dispersed in three 12 sunscreen formulations, in use under certain conditions were investigated in normal and 13

altered skin. Commercial products containing nano-sized particles of coated TiO2 and ZnO 14 dispersed in hydrophobic emulsions were used. One product contained only TiO2 (TiA), 15

another TiO2 plus ZnO (TiB) and a third material (TiHB) contained nanoparticles of coated 16 rutile form TiO2. 17

The nanoparticles were dispersed in hydrophobic basis gel composed by high pressure 18 polyethylene and viscous paraffin with Al2O3 (8-11%) and SiO2 (1-3%). The coated 19

preparations contained 76 – 82% TiO2. The size and shape of nanoparticles in the three 20

formulations were inspected with transmission electron microscopy and X-ray microanalysis. 21 Nanoparticles were needle-shaped and similar in both commercial and test formulation. The 22

application protocol consisted of an open test. The formulation was applied on the sacral 23 region and buttocks for 2 h, using a sunscreen application of approximately 0.5-1.0 mg/cm² 24

within an area of 25 cm². 25 The 3 formulations used in the study were tested in normal skin: TiB was applied to 9 26

individuals and both TiA and TiHB to 8 individuals. Nanoparticle penetration (TiA, TiB, TiHB) 27 was also evaluated in normal skin in an independent group of 10 individuals under non-28

physiological conditions induced by tape stripping and occlusive patches (48-hour 29

application). Tape stripping consisted of series of strips until the tapes were free of 30 corneocytes. A TiA-containing commercial sunscreen was further tested in involved skin 31

areas of 4 psoriatic patients. A matched control group constituted by 6 individuals was used 32 for the determination of basal elemental concentrations in skin including Ti. 33

Skin punch biopsies of 3 mm diameter were taken after application, quench-frozen and kept 34 in containers until processing. One biopsy was taken from each volunteer. Sections of 14 35

μm thickness were cut from the frozen biopsy in a cryostat at -25 °C. Biopsies were 36 mounted in mounting medium for microscopy. Sections were obtained from the non-37

immersed portion of the tissue, and sectioning performed from inside to outside to avoid 38

tissue contamination. Tissue integrity and the efficacy of corneocyte removal after tape 39 stripping were checked by preparing intercalary stained sections for optical microscopy 40

purposes. Scanning Transmission Ion Microscopy technique and Particle Induced X-ray 41 Emission technique were used for detection. The minimum detectable concentration of TiO2 42

in the skin was 0.31 μmol/g (24.8 μg/g tissue or 25 ppm). 43 44

Results 45 For imaging and localizing TiO2 and ZnO nanoparticles in intact skin, the coverage of the 46

outer skin layer with the TiA and TiB sunscreen formulations was homogeneously 47

distributed. The TiHB formulation showed a patchy distribution. Sunscreen formulations 48 accumulated in skin wrinkles and depressions as well as infundibulum cavities. Exogenous Ti 49

and Zn remained at the outer layers of the keratinized tissue that enfold the follicle i.e. 50 outside the living skin. 51

52 The nanoparticles penetration profiles obtained with the treated skin groups (TiA, TiB and 53

TiHB) were all similar. The high levels of TiO2 observed at the outer layers of stratum 54 corneum sharply decreased within deeper layers to become undetectable (as Ti by x-ray 55

emission technique). High Ti concentrations levels were only determined in the stratum 56

corneum of skin treated with the three formulations. In the subcorneal regions Ti 57

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40

concentration was below the minimum detectable concentration estimated for the analytical 1 technique. In non-treated skin Ti was below the minimum detection limit in all strata 2

inspected. For the depth positions, where TiO2 nanoparticle penetration ended an estimated 3 error of 10% was obtained, which approximately corresponds to 0.5 μm. In occluded skin, 4

there was no significant difference in TiO2 nanoparticles distribution and penetration depth 5 profiling. 6

7 Nanoparticle localization in damaged skin 8

Parts of the outer layers of the stratum corneum were removed by tape stripping (at least 9 15 strips) before sunscreen application. Removal of the stratum corneum was confirmed by 10

histological examination and ultimately by nuclear microprobe examination. Under this 11

condition there was negligible adhesion of the formulation tested (TiA). The TiO2 contents 12 determined on the skin outer layers were unimportant suggesting that, in normal skin, the 13

outer layers of stratum corneum trapped nanoparticles inside the desquamating corneocytes 14 network. 15

16 Results 17

In psoriatic skin, where the horny layer is thicker and less compacted than in normal skin 18 showed that the sunscreen formulation remained only, in the first layers of the stratum 19

corneum. The Ti distribution was often non-uniform and in some "hot-spots" sunscreen was 20

deposited at the outer layers of stratum corneum partly even in the hair follicle 21 infundibulum region. 22

23 Conclusion 24

The authors concluded that following 2 h exposure period of normal human skin to nano-25 sized TiO2-containing sunscreens, detectable amounts of these physical UV filters were only 26

present at the skin surface and in the upper most stratum corneum regions. Layers deeper 27 than the stratum corneum were devoid of TiO2, even after 48 h exposure to the sunscreen 28

under occlusion. Deposition of TiO2 and ZnO nanoparticles in the openings of the 29

pilosebaceous follicles was also observed. Penetration of nanoparticles into viable skin tissue 30 could not be detected. 31

32 SCCS Comments 33

The study is of good quality. Although for the TiO2 nanomaterial used in this study 34 information on surface area, number of particles per mass was not provided, the results 35

showed penetration of the nanoparticles only to the outer layers of Stratum corneum, but 36 not to the viable epidermis. The tested material relates to S75-N (>95% Rutile, <5% 37

anatase, coated with alumina 10% simethicone 2%, doped with 1000 ppm Fe). 38

39 Study Design: 40

Guideline/method: 41 Species: Porcine and human skin 42

Test substances: TiO2 uncoated nanoparticles, mixture of rutile and anatase, 43 average primary particle size 21 nm, uncoated, approximately 44

spherical platelets (Degussa-P25) 45 TiO2 coated nanoparticles, rutile, composition 76-82% TiO2, 8-46

11% Al2O3 and 1-3% SiO2, primary particle size about 20-100 47

nm, needle shaped (Eusolex T-2000, Merck KGaA) 48 Formulations: All formulations contained 5% TiO2 nanoparticles. 49

1. TiO2 uncoated: carbomergel, 20% propylenglycol, 0.5% 50 carbomer 500,000, 0.3% trometamol, and 79.2% purified 51

water. 52 2. TiO2 coated: hydrophobic basisgel, 5% high pressure 53

polyethylene and 95% viscous paraffin 54 3. TiO2 coated: polyacrylategel, 20% propylenglycol, 0.5% 55

carbopol 980, 0.3% trometamol, and 79.2% purified water. 56

Dose applied: 2 mg/cm² 57

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41

Skin: Porcine skin. The porcine skin specimens (n=12) were obtained 1 from domestic pigs. Specimens were sampled from the inner 2

parts of thighs in the form of punch biopsies. 3 Human skin. The human skin was obtained from the dorsal 4

region and buttocks of healthy adult volunteers (n=8). 5 Human grafted skin samples were produced from normal human 6

foreskins obtained from circumcision and grafted on a severe 7 combined immunodeficient (SCID) mouse model (n=4). 8

Skin temperature: Not stated 9 Exposure period: Porcine and human skin 2 h under semi-occlusive conditions, 10

human grafted skin 1 h, 24 h, and 48 h under occlusive 11

conditions. 12 GLP: No 13


Reference Gontier et al., 2008 (158) 16 17

18 Method 19

All three formulations were topically applied at 2 mg/cm2 and for 2 h to porcine and human 20

skin under semi-occlusive conditions, i.e., a breathable plaster protected the area. In a 21 previous pilot study with exposure times between 8 and 48 h no significant differences were 22

found for different exposure times. The sunscreen was applied to human skin grafted on 23 SCID mice for 1 h, 24 h, and 48 h under occlusive conditions. Untreated control samples 24

were also prepared for each analysis. 25 The skin biopsies (3 mm in diameter) were studied by Transmission Electron Microscopy 26

(HRTEM) and Scanning Transmission Ion Microscopy (STIM) combined with Rutherford 27 Backscattering Spectrometry (RBS) and Particle Induced X-Ray Emission (PIXE) on ultra-28

thin and thin cross-sections, respectively. 29

30 Results 31

Porcine skin 32 TiO2 uncoated. By superimposing the titanium distribution obtained by the PIXE map 33

on to the STIM map, it was possible to unambiguously determine the distribution of TiO2 34 particles via their chemical fingerprint with a close correlation to the epidermal layers. TiO2 35

particles were exclusively localized on the surface of the outermost SC layer. No titanium 36 could be found in the layers containing vital cells. The porcine skin after application of 37

hydrophobic basisgel exhibited a similar titanium distribution. To quantify the penetration 38

depth of TiO2 particles, a region of interest was chosen to extract the titanium depth profile 39 displayed. The extent of the profile was about 30 µm. A clear titanium peak is visible at the 40

skin surface, the titanium being strictly limited to the SC. The nuclear microprobe 41 observations were cross-checked by the results obtained on the same type of samples 42

studied by HRTEM. Apart from corneocyte layers, nanoparticles and agglomerates on and in 43 between the corneocytes are clearly visible. Electron X-ray microanalysis on individual 44

nanoparticles proved that they contain Ti. In addition, morphological features of the TiO2 45 particles were examined. The TiO2 particles sometimes appear as individual particles, but 46

more frequently agglomerated to clusters of different sizes. 47

48 TiO2 coated. An average size of 12 nm in width and of 60 nm for the length was estimated 49

for the primary needle-shaped particles. The large amount of the titanium particles for 50 both test emulsions, carbomergel and hydrophobic basisgel, was strictly located at the 51

surface of the last corneocyte layer with the possible exception of agglomerates below the 52 first and third corneocyte layer. 53

54 Human skin 55

The STIM map exhibits a thick SC and a well delineated SS containing keratinocyte cell 56

bodies. The Ti PIXE-maps are superimposed onto the STIM image, demonstrating that the 57

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42

particles were exclusively located on the outermost layers of the SC. This observation is 1 corroborated by the superimposition of the same titanium PIXE-map onto the RBS carbon 2

map. The depth profile of titanium extracted from the region of interest demonstrates that 3 the presence of this element is limited to a layer with a thickness of about 20 µm. 4

5 On the STIM map obtained for the commercial formulation, the SC is easily observable due 6

to its high density despite its unusually low thickness. In the titanium PIXE-map is 7 superimposed onto the STIM image. Ti is exclusively localized on the surface of the horny 8

layer. From the titanium depth profile, extracted from the region of interest, titanium was 9 found to penetrate into a 10 µm thickness layer of the SC only, but no titanium was 10

detected in the SS. 11

12 In the HRTEM micrograph, TiO2 particles were identified by the presence of large 13

homogeneous electron dense objects on the surface of the horny layer. At low magnification 14 the particles appear to be spread in a very homogeneous thin layer. With a high 15

magnification, the particles occasionally appeared as needle-shaped individual particles, but 16 most frequently aggregated in clusters of different sizes. The primary particles have a width 17

of 12 nm and an average length of 60 nm. Some particles were seen four to five layers 18 deeper, apparently only when a passage exists due to the looseness of corneocytes. 19

20

Human skin grafted to SCID-mice 21 The murine SCID model allows human skin to be grafted without any rejection. The 22

commercial product was applied for 2 h under occlusive conditions. Here, the STIM image 23 enables to delineate the SC from the large SS by its high density. In addition it shows the 24

papillary dermal-epidermal junction and the dermis. When the PIXE-titanium maps were 25 superimposed onto the STIM images obtained from the two different areas of interest a 26

microlesion, i.e., a partly detached horny layer, with Ti in the cleft was seen. The result 27 seemed to indicate that in some areas of the SC titanium penetrated more deeply compared 28

to other skin samples. The HRTEM micrographs revealed a thinner SC constituted by two or 29

three layers of corneocytes only. In fact, this sample was taken from the border between 30 mouse and human skin. The corneocytes are separated by larger spaces which have allowed 31

the product to penetrate down to the innermost corneocyte layer. The TiO2 particles seem 32 to be attached to the corneocyte layers. Nevertheless no TiO2 particles were observed in 33

the very close SG. 34 35

Conclusion 36 The authors concluded that whereas the HRTEM and STIM/PIXE images reveal clear 37

differences – mainly related to the different thickness of the cross-sections – they 38

unambiguously show that penetration of TiO2 nanoparticles is restricted to the topmost 3–5 39 corneocyte layers of the stratum corneum. 40

41 SCCS Comments 42

The study is of good quality. Although for the TiO2 nanomaterial used in this study 43 information on surface area, number of particles per mass was not provided, the results 44

showed penetration of the nanoparticles only to the outer layers of Stratum corneum, but 45 not to the viable epidermis. The tested material relates to S75-G (uncoated, anatase 85%, 46

rutile 15%), and S75-N (>95% Rutile, <5% anatase, coated with alumina 10% simethicone 47

2%, doped with 1000 ppm Fe). 48 49

50 Compromised skin 51

52 Study Design: 53

Guideline/method: Exploratory comparative percutaneous skin penetration study in vitro 54 after UVB radiation in vivo (sunburn simulation) 55

Test system: Skin of weanling Yorkshire pigs (approximately 20–30 kg) 56

Test substances: O/W and W/O sunscreen formulations 57

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A: T-Lite SF (coated, 10% O/W formulation, CM 630) 1 B: T-Lite SF (coated, 10% W/O formulation, CM 634) 2

CM 630 and CM 634 consist of TiO2 (rutile, crystallite of 14–16 nm) 3 coated with hydrated silica, dimethicone/methicone copolymer, and 4

aluminium hydroxide for a primary particle size of 10 x 50 nm and 5 specific surface area of 100 m2/g. The mean size of the agglomerates 6

was 200 nm with a range of ca. 90–460 nm 7 Batch: Not stated (source: BASF SE, Germany) 8

UVB exposure: A Fiber optic UVB lamp (Lightning cure 200 UV-Spot light) was used. 9 Reference: Monteiro-Riviere et al., (2011) (181, 182). 10

11

Method 12 On day 1 a pig was sedated and the hair clipped. The minimal erythemic dose (MED) was 13

determined by sequential exposure to UVB light (30 – 110 mJ/cm², - 22 sec.). On day 2 the 14 exposed sites were analyzed to determine the UVB dose required to produce 1 MED. The pig 15

was subsequently sedated and multiple sites (52 sites) on the back were exposed to the 16 UVB dose that caused a consistent +2 erythema, a pale red in a defined area of the skin. 17

Twenty-four hours after UVB exposure (Day 3), the pig was sedated, sites visually analyzed 18 for consistency, and the pig euthanatized. The UVB-exposed sites were dermatomed to a 19

thickness of approximately 400-500 μm and placed dermis side down on paper towels 20

saturated with physiological saline. 21 The skin prepared for the in vitro or in vivo studies. 22

UVB dose: 100, 110 and 120 mJ/cm² (pig 1, 2, 3 for MED of about 2.5) 23 24

In vitro part: 25 Dose level: 50 μl of each formulation on 0.64 cm² dermatomed pig skin 26

Skin preparation: Exposed and unexposed skin sites were dermatomed to 400 µm. 27 Dermatomed skin, placed dermis side down on towels saturated with 28

physiological saline, was cut into with a 19 mm circular punch. 29

Cells: Formulation A and B: 4 with UVB exposed skin, 2 with unexposed 30 skin 31

Control: 2 with UVB exposed skin, 2 with unexposed skin 32 Skin temperature: 37 °C 33

Test chamber: Flow-through diffusion cells 34 Route: Topical application 35

Exposure time: 24 hours 36 Sampling time 37

points: Every 2 h for the first 12 h, every 4 h thereafter up to 24 h 38

Examinations: Light microscopy (LM). Transmission electron microscopy (TEM) plus 39 X-ray microanalysis (EDS) Scanning electron microscopy (SEM). 40

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) 41 42

In vivo part: 43 Dose level: 250 μl of each formulation on exposed sites (n = 3 per formulation) 44

on 2 pigs on 1.0 cm² pad Hill Top chamber 45 Controls: Normal pig skin (no UVB, no sunscreen, no Hill Top chamber (n = 2 46

per pig) 47

UV-B exposed: No sunscreen, dry chamber (n = 2 per pig) 48 Sunscreen in a Hill 49

Top chamber: No UVB (n = 2 per formulation) 50 Route: Topical application 51

Exposure time: 2x 24 h and termination after 48 h 52 Sampling: Skin was removed by 8-mm biopsy punch 53

Examinations: As in vitro part 54 GLP: No 55

Published: Yes 56

57

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44

Method 1 The purpose of the study was to determine whether skin damaged by UVB radiation 2

inducing moderate sunburn with a +2 erythema reaction, enhanced the penetration of TiO2 3 or ZnO nanoparticles (see Opinion on ZnO (nanoform)) present in sunscreen formulations. 4

Weanling Yorkshire pigs (approximately 20–30 kg) were sedated and multiple sites (about 5 52) on the back were exposed to the UVB dose that caused a consistent +2 erythema (a 6

pale red in a defined area of the skin). 7 Twenty-four hours after UVB exposure, the pig was sedated, sites visually analyzed for 8

consistency, and the skin prepared for in vivo or in vitro studies. 9 For the in vitro studies, the UVB exposed and non exposed sites were dermatomed to a 10

thickness of approximately 400–500 μm. The dermatomed skin was mounted in the flow-11

through diffusion cells with a dosing area of 0.64 cm2 and maintained at 37°C. The skin was 12 equilibrated in perfusate and a flow rate of 2 ml/h for 30 min prior to dosing. The skin was 13

subsequently dosed with 50 μL of each formulation (CM 630: (n=4 UVB exposed skin, n=2 14 unexposed skin; CM 634: n=4 UVB exposed skin, n=2 unexposed skin; and control: n=2 15

UVB exposed skin, n=2 unexposed skin). After completion of dosing, the perfusion was 16 resumed and the perfusate collected every 2 hours for the first 12 hours and every 4 hours 17

thereafter up to 24 hours. After 24 hours, the perfusion was terminated and the skin was 18 removed from the diffusion cells. 19

20

The dose site was removed with an 8 mm biopsy punch and cut into thirds. One third was 21 placed in Trump’s fixative and stored at 4°C for later processing by light microscopy (LM; 22

flow-through 1 and 2 only) and transmission electron microscopy (TEM). The remaining 23 third of the skin was cut in half and immediately frozen and stored at -20°C for later 24

elemental analysis. The vials containing perfusate from each timed collection were capped 25 and the samples immediately stored at 4°C. 26

27 For in vivo treatment exposed sites (n = 3 per formulation) on two pigs were treated with 28

250 μl of each formulation; 200 μl was loaded onto the pad of the Hill Top chamber (1.0 29

cm² area) and 50 μl was placed directly on the skin within a template. Controls included 30 normal pig skin (no UVB, no sunscreen, no Hill Top chamber; n = 2 per pig), UVB-exposed 31

(no sunscreen, dry chamber; n = 2 per pig), and sunscreen in a Hill Top chamber (no UVB; 32 n = 2 per pig per formulation). Sites were redosed with new Hill Top chambers after 24 h, 33

and the treatment was terminated after 48 h. Erythema was scored for each site, and the 34 pigs were euthanatized as above. Skin from all of these sites was removed with an 8-mm 35

biopsy punch for microscopy studies as stated above. 36 37

Results 38

For the in vitro studies, light microscopy showed that UVB exposed skin showed focal 39 intracellular epidermal oedema, sunburn cells, dermal inflammation and focal microblister 40

and residual sunscreen containing TiO2 limited to the stratum corneum. The morphology of 41 the normal and the UVB-exposed skin was not affected by topical treatment with the 42

sunscreen formulations. The TiO2 in each formulation was confirmed by TEM and elemental 43 analysis. EDS found the presence of Ti and Cu (copper grid)) in CM 630 and CM 634. Si for 44

the coating, Pb for lead citrate and U for uranyl acetate staining. 45 In the in vitro flow-through studies, TEM/EDS found penetration of Ti to a depth of 9 layers 46

in the stratum corneum of normal skin and 17 layers in the stratum corneum of UVB-47

exposed skin. TEM/energy dispersive x-ray spectroscopy or inductively coupled plasma 48 mass spectrometry detected no Ti or Zn, indicating minimal transdermal absorption. 49

For in vivo tests, skin was dosed at 24 h occluded with formulations and at 48 h. TiO2 NP in 50 o/w formulation penetrated 13 layers into UVB-damaged SC, whereas only 7 layers in 51

normal skin; TiO2 in w/o penetrated deeper in UVB-damaged SC. Coated and uncoated ZnO 52 NP in o/w were localized to the upper one to two SC layers in all skin. TOF-SIMS showed Ti 53

within epidermis and superficial dermis, whereas Zn was limited to SC and upper epidermis 54 in both treatments. In summary, UVB-damaged skin slightly enhanced TiO2 NP or ZnO NP 55

penetration in sunscreen formulations but no transdermal absorption was detected. 56

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45

Conclusion 1 In summary, UVB-sunburned skin slightly enhanced the in vitro or in vivo penetration of the 2

TiO2 or ZnO NPs present in the sunscreen formulations into the stratum corneam (SC). 3 Although penetration of the two NPs into the SC was shown by TEM, and into the epidermis 4

and dermis by TOF-SIMS, there was no definitive evidence that they penetrated the skin in 5 vitro into the perfusate. In most cases, TiO2 penetration into the SC was greater than ZnO. 6

These results viewed together suggest minimal penetration of TiO2 and ZnO NPs into the 7 upper epidermal layers when applied topically in sunscreen formulations to normal and 8

UVB-sunburned skin, with no evidence of systemic absorption. 9 10

11

SCCS Comments 12 The study is of a good quality. The test material relates to S75-K (>94% rutile, coated with 13

6-8% aluminium hydroxide, 3.5-4.5% dimethicone/ methicone copolymer). The results of 14 transmission electron microscopy indicated penetration of TiO2 nanoparticles into stratum 15

corneum, whereas TOF-SIMS analysis indicated penetration into the epidermis and dermis. 16 However, analysis of perfusate by TEM/Energy Dispersive Analysis or ICP-MS did not detect 17

Ti or Zn indicating nanoparticles did not penetrate the skin in vitro. 18 19

In Vitro study (Senzui et al., 2010 - Ref 204) 20

21 Study Design: 22

Guideline/method: 23 Species: Yucatan micropig skin 24

Test substances: All TiO2 are rutile-type 25 T-35. size 35 nm, uncoated 26

TC-35, size 35 nm, coated alumina + silica + silicone 27 T-disp, size 10 x 100 nm, mixture of alumina coated and silicone 28

coated 29

T-250, size 250 nm, uncoated 30 Formulations: All formulations contained 10% TiO2 nanoparticles. 31

Cyclopentasiloxane (silicone, KF-995) used as dispersing medium) 32 Dose applied: 2 µl/cm² 33

Skin: Yucatan micropig skin removed the subdermal tissue and fat was 34 used as full-thickness skin (intact skin). The SC was removed from 35

intact skin with adhesive tape (Scotch 313, 3M) (stripped skin). Hair 36 was removed from intact skin using tweezers (hair removed skin) 37

Skin temperature: Not stated 38

Exposure period: 24 h 39 GLP: No 40


Reference: Senzui et al., 2010 (204) 43 44

Method 45 The TiO2 was suspended in a volatile silicone fluid used for cosmetics, cyclopentasiloxane, 46

at a concentration of 10%. The suspension was applied at a dose of 2 mg/cm2 for 24 h. 47

The skin penetration was investigated in vitro with intact skin and with stripped skin (the SC 48 removed from intact skin with adhesive tape) as a model of injured skin. In addition hair-49

removed skin (hair was removed from intact skin using tweezers) was used to represent 50 skin damaged by hair-removal treatment. 51

Two µl of suspension were applied to an area of skin of approximately 1 cm2. Then the skin 52 was placed on a modified Franz-type diffusion cell. After 24 h, the receptor phase (pH 7.1 53

isotonic phosphate buffer solution) was collected, the skin was removed from the diffusion 54 cell and cut off at the rim for mounting the cell. Residues on the skin surface were removed 55

by two cyanoacrylate stripping and Ti in the skin was determined. For some samples, the 56

epidermis and dermis were separated by heating after cyanoacrylate stripping. 57

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46

Skin conditions after application of TiO2 was observed using two methods. After application 1 and drying, the skin surface was observed by digital fine scope microscopy. The epidermis 2

of the skin prepared by a heat separation method was mounted on a scanning electron 3 microscope (SEM) stage with adhesive tape. 4

5 6

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47

Results 1 The particle size distribution of TiO2 in silicone was determined. The mean particle size of T-2

35 was 1700 nm, which was larger than that of T-250, 1200 nm. In contrast, suspensions of 3 the coated TC-35 and T-disp contained nanoparticles with mean diameter of 80 and 130 4

nm, respectively. 5 Ti concentration in the receptor phase was similar in all skin conditions and formulation 6

applied. For intact and stripped skin, no significant difference in Ti concentration was found 7 between the control and suspension applied, which indicates TiO2 did not penetrate into the 8

skin regardless of particles size and even when the SC was removed. For hair-removed skin, 9 Ti concentration in skin after application of TC-35 suspension was significantly higher than 10

that of the control, and after application of T-disp suspension, tended to be high. The Ti 11

concentration in the dermis was not different from the control. 12 Ti concentration in the epidermis after application of TiO2 nanoparticles tended to be 13

greater than that of the control, but the difference was not significant. The epidermis 14 consists of SC, viable epidermis and hair follicles. Ti was detected in the hair follicle pockets 15

of hair-removed skin, but not in the surrounding viable skin. The radius of a hair follicle is 16 0.05 – 0.2 mm which allow solvent to enter the hair shaft and sebum did not fill the follicle 17

space. When fluid enters a small space by capillary action, small particles of Ti in fluid may 18 be able to enter the follicle. Large particles cannot be moved by such small force, but TC-35 19

well dispersed in solvent might enter a follicle more easily than other types of TiO2. For T-20

disp, the dispersing agent had some effect, resulting in particles left in the skin after drying 21 of the suspension 22

23 Conclusion of the Applicant 24

The authors concluded that TiO2 does not penetrate into viable skin, even if the particle size 25 is less than 100 nm and the SC is damaged. However, immediately after hair removal the 26

concentration of Ti in skin was higher when TC-35 was applied, which was most probably 27 caused by dispersion. SEM-EDS observation showed that Ti penetrated into vacant hair 28

follicles but in any case did not penetrate into dermis or viable epidermis. It was noted that 29

since this was an in vitro study, inflammation could affect the results and further in vivo 30 studies on viable skin with hair removal are needed. 31

32 SCCS Comments 33

The quality of the study is difficult to evaluate. Moreover, the study was performed with skin 34 from Yucatan micropigs and experience with this skin type in skin absorbance studies is 35

limited. 36 37

38

In vitro exploratory study - percutaneous skin penetration - pig skin (Ref 70) 39 40

Study design. 41 Guideline/method: exploratory study 42

Species: pigs 43 Test substances: T805 (Degussa), hydrophobically coated with trimethyloctylsilane 44

Particle size: about 20 nm 45 Group sizes: n=2 skin samples 46

Dose applied: 0.8 mg total (20 mg with 4% TiO2), 0.16 mg TiO2 per cm2 47

Skin: fresh skin obtained from pigs used within 3 h after collection 48 Skin area 4.9 cm2 49

Skin temperature: 32oC 50 Test chamber: custom-made Franz-type diffusion cells 51

Receptor fluid: 0.9% w/v NaCl, 0.1% w/v gentamycin sulfate, 1% w/v bovine serum 52 albumin in bi-distilled water 53

Exposure period: 24 h 54 GLP: no 55

Published: yes 56

Study period: 1999 57

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Reference: Reference 70 submission III+IV Pflücker et al., 1999 1 2

Method 3 Fresh pig skin was obtained from the butcher, and used within 3 hours after collection. Skin 4

samples were punched (5 cm in diameter). The dermal absorption study was performed 5 with custom-made Franz-type diffusion cells. The lower cell was placed on a magnetic stirrer 6

(Variomag, Germany) and connected by tygon tubes to a thermostat (Type CS-C6, Lauda, 7 Germany) set at a temperature of 32°C (in vivo skin temperature). Magnetic stirring bars 8

were placed in the lower cells, which were filled with the receptor fluid (0.9% w/v NaCl, 9 0.1% w/v gentamycin sulfate, 1% w/v bovine serum albumin in bidistilled water). 20 mg of 10

the test emulsion, which contained 4% titanium dioxide, were topically applied with a 11

gloved finger to two excised pig skin discs (area 4.9 cm2, 2.5 cm in diameter, giving a 12 concentration of 4 mg cm2). After 24 h incubation 2 mm punch biopsies were obtained for 13

histological evaluation (TEM and SEM). SEM micrographs were recorded to evaluate the 14 morphology of the freeze-dried skin samples and the stripped stratum corneum sheets. 15

Freeze dried skin samples were investigated before and after 10–fold tape stripping. 16 17

Results 18 TiO2 was found exclusively on the outermost SC layer. No titanium dioxide could be found in 19

the living cell layers of the stratum granulosum. The surface deposit, as displayed by TEM, 20

featured clearly distinguishable agglomerates as well as single particles with a characteristic 21 cubic shape and a primary particle size of about 20–50 nm. Concurrently, SEM/EDXA 22

micrographs first showed an even distribution of TiO2 on the skin surface. After 10-fold 23 stripping, however, TiO2 was found to be localized only in the furrows and not on the 24

partially removed ridges of the skin surface. In the upper part of the hair follicle TiO2 was 25 demonstrated. 26

27 SCCS Comments 28

The actual TiO2 dose was 0.16 mg, and not 20 mg as mentioned in the paper. The study 29

does not show quantitative results but demonstrates by electron microscopy that the TiO2 30 nanoparticles are present on the skin mainly as aggregates. The study is of limited value 31

with number of samples investigated was only 2, but can be considered as supporting 32 evidence that TiO2 nanoparticles do not penetrate to the viable cell layers of the dermis. 33

34 In vitro exploratory study - percutaneous skin penetration and in vivo - human 35

skin (Ref 78) 36 37

Study design. 38

Guideline/method: exploratory study 39 Species: human healthy volunteers (female) 40

Test substances: Mixture of broad spectrum UV water-in –oil emulsions containing 41 water, glycerin, dimethicone, ethylhexyl methoxycinnamate, 42

isododecane, cyclomethicone, C12-15 alkyl benzoate, PEG-30 43 dipolyhydroxystearate, decyl glucoside, dodecyl glycol copolymer, 44

magnesium aluminium silicate, preservatives, zinc oxide, tocopheryl 45 acetate, o -cymen-5-ol, fragrance, xanthan gum and 3% ultrafine 46

TiO2 (T805, Degussa, Germany) and 8% methylene bis-47

benzotriazoyl tetramethylbutylphenol (MBBT) in a dispersion of decyl 48 glucoside. 49

TiO2 was coated with trimethyloctylsilane. 50 Particle size: TiO2 20 nm 51

Group sizes: n=3 52 Dose applied: 2 mg/cm2 of formulation, 60 μg TiO2 / cm

2 53

Skin: in vitro abdominal and face skin frozen until use, 54 in vivo skin of upper arm 55

Skin area in vivo 10 cm2 (2x5 cm) 56

Teflon static diffusion cell 10 cm2 (2x5 cm) 57

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49

Franz diffusion cell 1.13 cm2 1 Test chamber: Teflon® homemade static diffusion cell with a 10 cm2 (5x2 cm) 2

surface and a receptor volume of 8 ml. 3 Franz diffusion cell with a 1.13-cm2 surface and 5 ml of receptor fluid. 4

Receptor fluid: 0.9% NaCl water solution with 3% bovine serum albumin 5 Skin temperature: 32oC 6

Exposure period: 5 h 7 GLP: no 8

Published: yes 9 Study period: 2007 10

Reference: Reference 78 Submission VII Mavon et al., 2007. 11

12 13

Method 14 Samples of the mixture of broad spectrum UV water-in –oil emulsions were applied on skin 15

of volunteers (10 cm2, 2x5 cm) and on two types of diffusion chambers, one Teflon® 16 homemade static diffusion cell with a 10 cm2 surface allowing tape stripping of the test 17

system, and a Franz diffusion cell with a 1.13-cm2 surface. The applied dose for the in vitro 18 study was 60.6 ± 3.1 μg/cm2, and for the in vivo study 58.4 ± 1.9 μg/cm2. 19

The distribution of the sunscreens in the skin was directly assessed by the tape stripping 20

method, using adhesive tape (Scotch TM No. 6204, 3M Corp.). A total of 15 tape strippings 21 were applied onto the surface of the skin, and each was pressed on the skin 10 times with a 22

roller. Each strip was removed with 1 quick movement. No washing procedure was used. 23 The titanium analysis in the tape strippings and skin samples (epidermis, dermis and 24

receptor fluid) was based on a microwave assisted treatment, which digested the organic 25 components in the presence of sulphuric and nitric acid. The samples were then analyzed by 26

colorimetric assay, using diantipyrylmethane (0.5 g in 20 ml HCl 1 N). One ml of the colored 27 solution and 1 ml of the solution to be tested were mixed. The absorbance was read at 390 28

nm with a spectrophotometer (Anthelie Advanced, France) 30 min later. Using this 29

technique, a LOD of 0.2 μg/ml was obtained. 30 Transmission electron microscopy and particle-induced X-ray emission (PIXE) techniques 31

were used to localize the TiO 2 in skin sections. Punch biopsies of 6 mm in diameter were 32 made on skin samples, consecutively after 1, 8 and 15 tape strippings and were fixed with 33

2% glutaraldehyde in a Sorensen buffer for TEM analysis. 34 35

Results 36 For the in vitro experiments with n=3 >94.2% of the recovered TiO2 was found in the 15 37

tape strippings and in the stratum corneum. In the epidermis 5.6% was found, and <0.1% 38

was found in the dermal compartment. No TiO2 was found in the receptor fluid (below LOD). 39 The amount recovered accounted for 88.8% of the applied dose of TiO2. In the in vivo study 40

(n=3) the recovery was 93% of the TiO2 dose. Most of the recovered dose was in the first 41 three tape strippings. After 15 tape strippings a few grains could be distinguished in the 42

TEM samples (amplification x 15,000), attributed to TiO 2 nanoparticles, but they were very 43 few and isolated in the stratum corneum (SC) layer. Deeper in the SC, no particles could be 44

observed, which suggested an absence of penetration into the viable skin tissue. 45 The 2-dimensional mapping of titanium using Micro-PIXE analysis of the skin showed that 46

most of the Ti applied at the skin surface remained there or penetrated only into the opened 47

infundibulum. Quantitative analysis revealed a concentration of Ti at the LOD, in the 48 underlying layer of the epidermis, the dermis, the follicle and the sebaceous glands. 49

It was concluded by the authors that the study confirms that TiO2 accumulates in the 50 uppermost layers of the SC and in the opened infundibulum only. No TiO2 was detected in 51

the viable skin layers through either transcorneal or transfollicular pathways. From these 52 data the authors concluded that the amount of TiO 2 found in the in vitro ‘epidermal’ 53

compartment is located mainly in the furrows or the opened infundibulum and does not 54 represent actual transcorneal penetration. 55

56

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SCCS Comments 1 Both TiO2 and MBBT were present in the broad spectrum UV water-in–oil emulsions. Lack of 2

penetration of TiO2 was supported by both in vitro and in vivo studies. Whether the detected 3 particles were attributed to TiO2 or not has not been identified by the study. 4

5 6

In vitro study - Percutaneous skin penetration pig skin (Ref 56) 7 8

Study design. 9 Guideline/method: yes (OECD 428, SCCNFP/0750/03) skin absorption in vitro method 10

Species: pig 11

Test substances: T-Lite SF-S coated with silica (2%-5% wt%) and methicone (4.5%-12 6.5%) 13

T-Lite SF coated with methicone (3.5%-5.5%) 14 Particle size: T-Lite SF-S, needle like with a size of 30-60x10 nm 15

T-Lite SF, needle like with a size of 30-60x10 nm 16 Both TiO2 materials were present including aggregates up to 200 nm 17

and higher (1 μm) 18 Group sizes: skin from 3 pigs, and per sample 3 skin preparations (n=9) 19

Dose applied: 4mg/cm2 corresponding to nominal doses of about 400 μg/cm2 of 20

titanium dioxide or to nominal doses of 21 240 μg/cm2 of titanium, 22

Skin: full thickness skin samples from lateral abdominal region 23 Skin area about 1 cm2 24

Skin temperature: 32 ± 1oC 25 Test chamber: modified Franz static dermal penetration cells 26

Receptor fluid: physiological saline containing 5% bovine serum albumin 27 Exposure period: 24 h, sampling at various time intervals (3, 6, 12, and 24 h) 28

GLP: yes 29


Reference: Reference 56 Submission VII Gamer et al., 2007 32 33

Method 34 After removal of the receptor fluid the skin was removed from the diffusion cell and put onto 35

parafilm. Titanium was removed from the skin preparations by washings with sponge pieces 36 dipped into soap solution, and subsequent tape stripping was used to remove titanium 37

together with the superficial layers of the stratum corneum. Ti was determined by 38

inductively coupled plasma-atomic emission spectrometry (ICP-AES) or ICP-mass 39 spectrometry (ICP-MS). 40

41 Results 42

For the titanium dioxide formulations T-Lite SF-S and Tlite SF, mean total recoveries of Ti 43 ranged from 98% to 100% and 86% to 93% of the total Ti applied, respectively. Virtually 44

the total amount of applied Ti could be removed from the skin surface by washing. The 45 amounts of titanium found in the tape strips and skin preparations were in the order of the 46

analytical determination limit. No Ti was found in the receptor fluid at any sampling time. 47

48 SCCS Comments 49

This is a GLP study with three independent measurements indicating lack of TiO2 50 penetration in an in vitro assay using pig skin. Although the number of measurements 51

(n=3) per skin is limited, it was repeated in skin samples of three different pigs. 52 53

54 55

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In vitro exploratory study percutaneous skin penetration, and in vivo study on 1 human skin (Ref 130) 2

3 Study design. 4

Guideline/method: exploratory study 5 Species: human (male and female) 6

Test substances: commercial microfine TiO2 dispersion either in octyl palmitate or in 7 water (Tioxide Specialities Ltd) 8

Particle size: not reported 9 Group sizes: n=3 10

Dose applied: in vitro 150 μl/cm2 of commercial preparation (5% TiO2, 7.5mg/cm2) 11

in aqueous or oily dispersion 12 In vivo 2 μl/cm2 (5% TiO2, 0.1 mg/cm2) in aqueous or oily dispersion 13

Skin: in vitro human skin from abdominal area (samples stirred at -20oC), 14 and skin equivalents with cultivated human keratinocytes and 15

fibroblasts 16 In vivo ventral side of forearm of male and female volunteers 17

Skin area not reported 18 Skin temperature: 32oC 19

Test chamber: penetration cells identified with figure. 20

Receptor fluid: phosphate buffer pH 7.4 21 Exposure period: 24 h for in vitro studies 22

45 minutes for the in vivo studies 23 GLP: no 24


Reference: Reference 130 Submission VII Bennat and Müller-Goymann 2000 27 28

Method 29

A penetration cell was used for both skin samples and the human skin equivalent studies. 30 For in vitro test the amount added was 150 μl per skin sample, for the in vivo tests 2 μl per 31

skin area. This results in TiO2 administrations of 7.5 mg/cm2 and 0.1 mg/cm2, respectively. 32 All dispersions were removed after the exposure period (in vitro 24 h, in vivo 45 minutes) 33

with a paper towel. Both in vivo and in vitro Tesa® were used for collection of cell layers of 34 the skin treated with the TiO2 formulations. Atomic absorption spectrometry (AAS) was used 35

for determination of the Ti content. Tests were performed in triplicate. The formulations 36 investigated were: an oil/water emulsion with carboxymethylcellulose (CMC), and 37

dimethicon and silicon oil; a liposomal formulation with phospholipid and water. 38

39 Results 40

The amounts of Ti observed after the in vitro and in vivo exposure of skin was in the μg 41 range. In the sequential tape strips starting at about 25-35 μg/cm2 in the first tape strip 42

and declining just above the limit of detection (0.1 μg/cm2) level at tape strip #6-#12. For 43 the oily dispersion having the highest Ti levels were measured in the first tape strips. For 44

the in vivo exposure the Ti recovery started at about 7.5 μg/cm2 and declined in the 45 following tape strips. Microfine TiO2 was found to penetrate deeper in the human skin from 46

an oily dispersion than from an aqueous one. 47

48 SCCS Comments 49

No information was provided on the actual size of the used microfine TiO2, so the study can 50 only be considered as supporting evidence. 51

52 53

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In vitro exploratory study percutaneous skin penetration - human skin (Ref 142) 1 2


Species: human (female Caucasian) 5 Test substances: Solaveil CT10W 3% W/Si emulsion, 3% W/O emulsion, both used as 6

sprayable product (Uniqema, UK) 7 Particle size: not reported in M&M section. Mentioned in Discussion to be between 8

20-70 nm 9 Group sizes: n=6 (experiments) 10

Dose applied: 2 mg/cm2 11

Skin: abdominal skin from plastic surgery stored at -25oC for maximally 6 12 months 13

Skin area 5.31 cm2 14 Skin temperature: 32 ± 1 oC 15

Test chamber: static Franz-type diffusion cell, 16 Receptor fluid: PBS with 4% bovine serum albumin 17

Exposure period: 1, 2, 4, 6, 8, 12 and 24 h 18 GLP: no 19

Published: yes 20

Study period: 2009 21 Reference: Reference 142 submission VII Durand et al., 2009 22

23 Method 24

The incubation in the diffusion cells was performed in wells covered with Parafilm paper to 25 avoid drying, and the whole system was protected from sunlight by opaque paper. Receptor 26

fluid was removed at several time points and replaced immediately with fresh solution. Ti 27 level was quantified and determined by Inductively Coupled Plasma-Optical Emission 28

Spectroscopy (ICP-OES). Samples were digested and dissolved before Ti determination. 29

At the end of the experiment, the skin samples were removed from the cell and rinsed with 30 PBS solution and tetrahydrofuran/ acetonitrile (THF/CAN, 80 : 20, v : v) until no product 31

was left on the skin. The skin was then ground and mixed with a THF/ACN (80 : 20, v : v) 32 solution and placed in an ultrasonic bath for 30 min. Each solution was then divided in two 33

parts: one part was kept at -25oC for the further analysis of the TiO2 by spectrometric 34 methods. Three types of sample were taken and analysed: 35

1 The receptor fluid (5 mL). 36 2 A solution of the recovered product remaining on the skin (after evaporation of all liquid). 37

3 The mixture of ground skin (after evaporation of all the liquid). 38

The samples were heated at 450oC in a muffle furnace for 10–12 h. They were then fused 39 with 5 g of K2S2O7 in a flame. The resulting substance was dissolved in 10 mL hot H2SO4 40

solution (1 : 1 v/v) and diluted with ultra-pure water to 100 mL. 41 The solutions obtained were then injected into the ICP-OES apparatus. 42

43 Results 44

The recovery of the TiO2 from the emulsions and spiked PBS solution with 4% BSA was 45 92.5% (W/O emulsion), 92.4% (W/Si emulsion, and 96.8% for the BSA-PBS solution , 46

respectively, demonstrating the validity of the method for determination of Ti. In each part 47

of the skin and in the receptor fluid for W/O and W/Si, respectively. the levels of recovery 48 were between 76% and 86% for Ti present in the skin and/or on the skin. The limits of 49

detection and of quantification are respectively 0.01 ppm (0.01 μg/g) and 0.1 ppm (0.1 50 μg/g) for the W/O and W/Si emulsion. Presence on skin after washing was about 40% and 51

50% for the W/O and W/Si emulsion, respectively. Approximately 20% (W/O) to 40% 52 (W/Si) of the TiO2 was observed in the skin. After 24 h of experiment titanium levels were 53

below the limit of detection. So it was considered that no TiO2 passed into the receptor fluid. 54 There was a loss in the recovery up to 25% of the administered dose. 55

56

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SCCS Comment 1 No characterization data for TiO2 is presented - only size has been indicated in discussion 2

section of the paper. Presence of coating indicated in Table 4.2 of supplicant but not 3 mentioned in the paper. Data on receptor fluid indicated in text but not shown in paper. 4

Level of Ti at 24 h mentioned to be below limit of detection but data on 5 recovery/determinations at various time points are not presented in the paper. Results are 6

of limited value for the evaluation of skin penetration of TiO2 as no data on the receptor 7 fluid were presented. It was demonstrated that approximately 20% to 40% of the TiO2 was 8

observed in the skin. No further evaluation of localization was done. 9 10

11

In vitro exploratory study percutaneous skin penetration - human skin (Ref 143) 12 13


Species: human 16 Test substances: Titanium dioxide T805, and Spectra veil MOTG, a 60% dispersion of 17

zinc oxide in mineral oil/triglyceride. 18 Particle size: not reported 19

Group sizes: not reported 20

Dose applied: 1 mg/cm2 in vitro 21 Skin: abdominal skin recovered from plastic surgery 22

Skin area not reported 23 Skin temperature: room temperature 24

Test chamber: not reported 25 Receptor fluid: not reported 26

Exposure period: not reported 27 GLP: no 28

Published: yes 29

Study period: 1997 30 Reference: Reference 143 submission VII Dussert et al., 1997. 31

32 Method 33

The presence of TiO2 in the skin was evaluated by TEM. At TEM characterization the TiO2 34 was identified as a mixture of rutile and anatase crystal forms. The sunscreen formulation 35

investigated was a mixture of both TiO2 and ZnO. The test formulation was a w/o emulsion 36 formulated with ultrafine titanium dioxide (11% wt), and zinc oxide (2.5% wt). The 37

formulation was used as topical administration in vitro with a dose of 1 mg/cm2 Skin 38

penetration was evaluated by TEM. 39 40

Results 41 Cross-sections of the horny layer of human epidermis, after topical application of the 42

sunscreen emulsion, show an almost regular mineral-coating of the stratum corneum. The 43 crystals appear to surround the desquamating corneocytes. However, neither intercellular 44

nor intracellular penetration of crystallites is evident in transmission electron microscopy. 45 The TEM evaluation shows the presence of particles above the stratum corneum and 46

between desquamating stratum corneum cells. 47

48 SCCS Comments 49

Although this study provides some evidence that there is no penetration of the 50 nanoparticles from the formulation into the skin, the information on the study itself is rather 51

limited, e.g. time of incubation and surface area of treated skin were not indicated. A 52 mixture of TiO2 and ZnO nanoparticles was used in the formulation. In the TEM evaluations 53

the TiO2 and ZnO could not be identified separately. This study is of no value for the 54 evaluation of skin penetration of TiO2 nanoparticles. Presence of coating is indicated in Table 55

4.2 of supplicant but not mentioned in the paper. 56

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Kertesz et al. 2004, Ion-microscopic evaluation of porcine or human skin after 1 treatment with TiO2 samples (Ref 66) 2

Samples investigated by ion microscopy are 14-16 µm thick porcine and human skin. 3

Quantitative elemental concentrations and distributions a new measurement setup and data 4

evaluation system has been developed. 5

The penetration studies using different formulations were started on domestic pig skin, 6

which resembles human skin closest. In a next step, human skin xenografts transplanted 7 into SCID mice were used. 8

22 pig skin, 11 transplanted human skin and 13 human skin samples were investigated. 9

Results 10

The results obtained by ion microscopy or electron microscopy show that in the case of 11

healthy skin the nanoparticles penetrate into the deepest corneocyte layer of the skin, but 12 never reach the vital layers. 13

14 Conclusion 15

No penetration of the test material into viable porcine or human skin 16 17

Nanoderm - Quality of skin as a barrier to ultra-fine particles (ref 67) 18

Penetration of TiO2-nanoparticles through the epidermis of human foreskin grafts 19

transplanted into SCID (Severe Combined Immune Deficiency) mice. 20

The skin grafts were treated with a hydrophobic emulsion (Antheil’s XL F60) containing 21 micronized TiO2-nanoparticles in occlusion, for 1, 24 and 48 h. 22

Quantitative elemental concentrations and distributions have been determined in 14-16 µm 23 thick freeze-dried sections obtained from quick frozen punch biopsies using PIXE (Particle 24

Induced X-ray Emission), STIM (Scanning Transmission Ion Microscopy) and RBS 25 (Rutherford Backscattering) analytical methods. 26

Result 27 In most cases it was found that the remnant of the liposome crème together with the 28

outermost stratum corneum was removed during the sample preparation. When the crème 29

remained on the skin the Ti was quasi homogeneously distributed in the outermost layers, 30 and the penetration seemed to be limited to the outermost part of the stratum corneum. 31

However, in two cases, both after 48 h exposure, penetration through the stratum corneum 32 to the limit of the vital stratum granulosum was observed. The sample originates from the 33

entry of a sweat gland. 34 35

Conclusions 36 No penetration to the viable skin was reported except for some limited observations of 37

material entering sweat glands. 38

39

Adachi et al., 2010, In vivo effect of industrial titanium dioxide nanoparticles 40

experimentally exposed to hairless rat skin (Ref 126) 41

Guideline/method: No specific guidelines followed 42

Test system: Hairless Rat (Male Westar Yogi Rats) 8 weeks old, weighing 202–267 43 g, (Japan SLC, Hamamatsu) 44

Test items: Uncoated anatase TiO2 nanoparticles (ST-01) from Ishihara Sangyo, 45 Ltd, Japan. 46

Formulation White water/oil (W/O) emulsion containing 10 wt% TiO2, 4 wt% 47

Nikko Nikkomulese WO (cyclopentasiloxane, PEG-10 dimethicone, 48 dosteardionium hectrite), 50.0 wt% decamethylcyslopentasiloxane 49

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KF-995 and 0.55 wt% acetic acid, and purified water was added to a 1 final volume of 100 wt% 2

Concentrations: Four mg/cm2 emulsion (0.4 mg/cm2 TiO2) was applied to a 15 cm2 3 area on the rat dorsal skin in the absence of ultraviolet (UV) 4

radiation. 5

Exposure: Skin samples at 4 h after exposure were observed using light, 6

electron, and confocal laser scanning microscopy over 48 hrs. Time 7 course study for light microscopic evaluation in the other groups of 8

rats (10 TiO2-treated and five control rats) was carried out at 24, 72 9 and 168 h after exposure. 10

Results 11

After 24 h, no particles were observed in keratinized layers of the follicular infundibulum, 12 but a small amount of particles remained in the superficial part of the stratum disjunctum. 13

After 72 h, the particles were still observed in upper keratinized layers of the infundibulum 14 but were not found in the interfollicular horny cell layer (Figure 3d). After 168 h, small crops 15

of particles were found in the uppermost keratinized layer of only a few follicular openings. 16 17

Conclusion 18 The study shows no penetration of TiO2 in water / oil emulsion into viable skin through 19

either the transcorneal or transfollicular pathway. 20

21

Gopee et al., 2009, Lack of dermal penetration following topical application of 22

coated and uncoated nano- and micron-sized titanium dioxide to intact and 23 dermabraded skin in mice (Ref 162 - poster presentation) 24

Guideline/method: No 25

Test system: Mice (hairless) 26

Test item: TiO2 (Unreported batch) roughly spherical uncoated particles, 27 with 25.1 ± 8.2 nm diameter (minimum particle size was 13 nm 28

and maximum particle size was 71 nm). Formulation consisted 29

of titanium dioxide suspended in polyglyceryl-3 distearate, 30 cetearyl alcohol, light mineral oil, propylene glycol, k-phosphate 31

buffer, methyl paraben, propyl paraben, and propylene 32 glycol:water (1:4, v:v). 33

Treatment: Mice (hairless) were treated with 5 uL of 5% uncoated anatase 34 TiO2 (intact or dermabraded skin). At 6 and 24 hr post-35

application, mice were sacrificed and skin, right regional lymph 36 nodes, blood, liver, kidney and spleen were collected and 37

analyzed for titanium (Ti) by ICP-MS. Tissues of one mouse was 38

analyzed microscopically. 39

Result 40

No significant elevations in Ti levels were observed in any of the organs analyzed for Ti. 41 42

Conclusion 43 The results suggest that both intact and compromised skin of hairless mice may be an 44

effective barrier for nano-sized TiO2. 45 46

Kiss et al. 2008, Investigation of micronized titanium dioxide penetration in 47

human skin xenografts and its effect on cellular functions of human skin-derived 48 cells (Ref 167) 49


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Test system: In vivo SCID mice, grafts area, 6-mm diameter human foreskin 1 punch biopsies were taken. 2

In vitro: human immortalized HaCaT keratinocyte cells, human dermal 3 fibroblasts (HDFs) & human immortalized sebaceous gland cell 4

line SZ95. 5

Test items: TiO2, 9 nm Anatase (gift from Prof. Z. Stachura, Krakow, 6

Poland) 7

Vehicle: hydrophobic emulsion (‘TiO2-emulsion’) was used (Anthelios XL 8

SPF 60, La Roche Posay, La Roche Posay, France) 9

Concentrations: 2 mg / cm³ 10

Exposure: 24 h 11

Result 12 TiO2 particles did not penetrate through the stratum corneum of human skin transplants. 13

TiO2 nanoparticles are internalized by in vitro cultured fibroblasts and melanocytes but not 14 by keratinocytes and sebocytes. 15

16 Conclusions 17

This type of TiO2 (custom made, anatase) does not penetrate human foreskin grafts. In 18 vitro uptake is cell type dependent. 19

20

Pinheiro et al. 2007, The influence of corneocyte structure on the interpretation of 21 permeation profiles of nanoparticles across skin (Ref 191) 22


Test system: Healthy and psoriatic human skin was collected by .punch 24

biopsy (3 mm diameter) at lumbar-sacral region, 25

Test material: Commercial sunscreen formulation (unknown source), 26

containing nano TiO2. 27

Concentrations: Unknown 28

Exposure: 2h 29

Results 30 The TiO2 permeation in psoriatic skin reached deeper regions of the stratum corneum than 31

in healthy skin. However, for both cases TiO2 nanoparticles did not reach the living layers of 32 the granulosa or spinosum strata. 33

34

Conclusion 35

Psoriasis seems to have only a limited effect on the permeation profile of TiO2 36 nanoparticles. It has to be mentioned that the source and concentration of the particles is 37

not specified in this study. 38

39

Popov et al. 2005, 2005, 2010 (Ref 192, 193, 194) 40

Test system: human skin (volunteers). Sunscreen including rutile TiO2 41 particles (100 nm) was administered five times over a period of 42

4 days onto the surface area of flexor forearm skin. The tape-43 stripping procedure started on the fourth day, 1 h after last 44

application. The surface density of TiO2 particles on the tape 45 strips was analyzed by x-ray fluorescent measurements. 46

Test material: Sunscreen including rutile TiO2 particles (100 nm), this was not 47

further specified. 48

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Concentration: 2 mg/cm² sunscreen. skin area of 10 X 8 cm (160 mg 1 sunscreen). 2

Results 3 Approximately 14 µg/cm2 of TiO2 was found on the first tape strip and almost zero on those 4

taken at the depth of 15 µm. The particles were mainly located at a depth range of 0 to 3 5 µm. 6

7 Conclusions 8

No penetration into living layer of skin. The source and nature of TiO2 is not well reported. 9 Three different papers all presenting the same experiment as an original study. 10

11

Sadrieh et al. 2010, Lack of significant dermal penetration of titanium dioxide 12 (TiO2) from sunscreen formulations containing nano- and sub-micron-size TiO2 13

particles (Ref 199) 14

Test system: Female Yucatan minipigs (~4 months of age; n ¼ 12) from 15

Sinclair Research Center (Auxvasse, MO, USA). 16

Test items: Uncoated nano titanium dioxide (Degussa Aeroxide P25, a 17

mixture of anatase and rutile and known to be photocatalytic; 18

1. coated (aluminum hydroxide/dimethicone copolymer) nano 19

titanium dioxide (BASF T-Lite SF obtained from BASF, 20

Shreveport, LA; rutile; ‘‘coated nano’’) 21 2. uncoated submicron titanium dioxide (treated with 22

aluminum hydroxide, Ishihara Tipaque CR-50 obtained from 23 Ishihara Corporation, San Francisco, CA; rutile; 24

‘‘submicron’’) 25 Vehicle All used particles were added to the same sunscreen 26

preparation, preparation without particles was used as control. 27

Concentrations: Approximately 5% preparations were achieved. 28

Exposure: Topical application four times daily, 5 days a week, for a total of 29

22 days. Dose of 2 mg/cm², each animal received a total of 176 30 mg/cm² cream resulting in a average of ~1.32 l of cream per 31

animal 32

Negative control: cream without TiO2 33

Result 34 The epidermis from minipigs treated with sunscreens containing TiO2 showed elevated 35

titanium levels. Increased titanium was detected in abdominal and neck dermis of minipigs 36 treated with uncoated and coated nano TiO2. EM-energy dispersive x-ray analysis showed 37

that TiO2 particles were found in the stratum corneum and upper follicular lumens in all 38

treated skin samples. Isolated titanium particles were present at various locations in the 39 dermis of animals treated with any of the three types of TiO2 sunscreens; however, there 40

was no pattern of distribution or pathology. 41 42

Conclusion 43 These findings indicate that there is some, though probably not significant, penetration of 44

TiO2 nanoparticles through the intact normal epidermis in minipigs. The quantification of the 45 concentration in the dermis is difficult since the removal of the epidermis is almost never 46

perfect (resulting in possible false positive results). 47

48 Exploratory study, dermal penetration and toxicity, hairless mice and porcine skin, 49

subchronic dermal exposure (Wu et al., 2009) 50 51

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The paper has its focus on the penetration of TiO2 nanoparticles through the skin after 1 dermal exposure. 2

3

No penetration in in vitro porcine skin model of TiO2 (4, 10, 25, 60 and 90nm). The 4 amount of TiO2 was below detection limit, but materials and methods stated that TiO2 5

was not removed. Not clear whether the TiO2 was removed before tape stripping. 6

Results indicate that tape stripping most probably was done after removal of TiO2, 7 hence there was a low levels in the tape strip pools. 8

Pig skin in vivo: TiO2 present in stratum corneum, stratum granulosum, prickle cell layer 9 and stratum basale of the epidermis but not in dermis. Only 4nm TiO2 in basal cell 10

layer. Figure 2 does NOT clearly show presence of TiO2 nanoparticles in epidermis. 11

Hairless mice: Effect of TiO2 on body weight observed. Decreased growth compared to 12 control mice and mice treated with normal sized TiO2. 10-25- and 21 (P25) nm TO2 13

induced growth retardation. 14

Biochemical parameters for skin and liver malondialdehyde (MDA) increase (10-25-15 21nm), superoxide dismutase (SOD) skin and liver decrease (10-21nm), skin 16 hydroxyproline (HYP) decrease (10-25-21-60nm) 17

Organ distribution after 60 days skin exposure showed 10, 25, 21, 60nm TiO2 in skin, 18 sub muscles, heart, liver, spleen, 21, 60nm TiO2 in lung, 21nm TiO2 in brain, whereas 19

TiO2 in kidney was similar to control. However the differences were not significant. 20

21

Conclusion 22 Local effects on skin are demonstrated by biochemical parameters SOD, MDA, and HYP, and 23

histopathology (keratinization). Systemic effects are not clearly identified because of 24

possible alternative route of exposure by oral uptake. Also the lesions shown in various 25 organs may be due to background lesions present in animal strain. This is not excluded by 26

scoring of lesions in control versus treated animals. However, the treatment resulted in 27 growth retardation of the animals. 28

29 Studies with limited information 30

31 In vivo study (Gottbrath et al., 2003; FitzGeral, 2005) 32

Penetration of nano-sized titanium dioxide (Tioveil AQ N; (rutile, coated with alumina/silica) 33

into human stratum corneum after in vivo application of two formulations was studied. 34 Penetration was measured by tape stripping of skin (10 strips). Tape strips from the 35

titanium dioxide-treated skin sites were assayed for titanium by atomic absorption 36 spectrometry. Tape strips from the vehicle control treated sites were viewed with an 37

inverted microscope to estimate the amount of corneocyte aggregates. Titanium dioxide 38 nanoparticles in the formulations and tape strips were visualized by transmission electron 39

microscopy (TEM). The authors concluded that, after application of the liposomal 40 formulation, a fraction of the TiO2 nanoparticles penetrated into the stratum corneum and 41

did not remain in shallow valleys formed by the corneocytes, explaining the water resistance 42

of the liposomal formulation, i.e. the deposition of TiO2 nanoparticles depends on the 43 formulation used. 44

45 In vivo study (Tan et al., 1996; FitzGeral, 2005) 46

Review of recent literature on safety of nanomaterials in cosmetics with special references 47 to skin absorption and resorption of ultrafine titanium dioxide and zinc oxide, prepared for 48

Physical Sunscreens Manufacturers Association (PSMA), European Cosmetic, Toiletry and 49 Perfumery Association and BASF AG, 28 September 2005. 50

51

A study with 10-50 nm TiO2 particles was performed in order to evaluate if the particles 52 could penetrate the stratum corneum to the dermis following repeated application in 53

volunteers (13 patients with compromised skin scheduled to have surgery for skin lesions). 54

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The patients received repeated application (twice a day for 2-6 weeks) of a sunscreen lotion 1 containing 8% microfine TiO2. Chemical analysis (ICPMS) were performed on skin biopsies. 2

The authors concluded that non-statistically significantly higher Ti levels in the dermis of 3 treated subject vs. controls (cadaver skin) were found. 4

5 In vivo study (Lademann et al., 1999) 6

The dermal penetration of 20 nm TiO2 nanoparticles (Titan M 160, coated, rutile) (assumed 7 particle size, based on description of product used) in a sunscreen formulation (o/w 8

emulsion) was studied. The sunscreen was applied repeatedly (11 times) over 4 days to the 9 forearm skin (2 mg/cm2) of human volunteers. UV/Vis spectroscopic evaluation, X-ray 10

fluorescence measurements LIFM, SRLSM and Raman spectroscopy of skin tape strips and 11

histological evaluation of skin biopsies were performed. The only significant finding 12 concerning a potential penetration of TiO2 beyond the upper skin layers was their deposition 13

in single hair follicle openings, although there was no evidence that these residues were 14 located within the living skin. The concentration of Ti in the hair follicle openings was two 15

orders of magnitude lower than that in the upper skin layers. The authors concluded that 16 that there was no penetration of TiO2 particles in living skin and that the TiO2 particles 17

were mainly located in the outer layers of the SC. 18 19

In vivo study (Schulz et al., 2002) 20

The influence of particle size on the dermal absorption of three TiO2 preparations was 21 investigated (T805 [20 nm, cubic, Ti/Si coating, rutile/anatase], Eusolex T-2000 [rutile, 10- 22

15 nm NPs in 100 nm aggregates, needles, Ti/ Al2O3/SiO2 coated] Tioveil AQ-1 0P [100 23 nm, needles, Ti/Al/Si coated]). Each had a different primary particle size (10-15 nm, 20 nm 24

and 100 nm), shape (cubic or needles) and hydrophobic/hydrophilic characteristics. The 25 preparations were topically applied (4 mg/cm2) in an oil-in water emulsion containing 4% 26

TiO2 to the forearm skin of human volunteers for 6 hours. Skin biopsies were examined by 27 scanning electron microscopy to visualize the distribution of particles within the skin layers. 28

TiO2 particles were only deposited on the outermost surface of the SC, and were not 29

detected in deeper SC layers, the human epidermis and dermis. The authors concluded that 30 none of the particles penetrated beyond the outer layer of the stratum corneum. 31

32 Another study provided under dermal penetration (Reference 10, submission 1) seems to be 33

an irritation study and has therefore not been reviewed. 34 35

SCCS Comments on Dermal/ Percutaneous Absorption 36 The studies presented in the submission cover a range of nanomaterials of which some 37

relate to the materials under assessment. The studies range from in vitro to ex vivo and in 38

vivo experimental conditions, and intact and UV damaged skin. The results from these 39 studies suggest that TiO2 nanoparticles, when applied to skin in a sunscreen formulation, 40

are likely to stay largely on the skin, whilst a small proportion of the particles may 41 penetrate to the outer layers of stratum corneum. A few reports have suggested the 42

possibility that TiO2 nanoparticles may penetrate deeper to reach stratum granulosum – 43 e.g. in human foreskin grafts transplanted onto SCID mice (Kertész et al., 2005) - or to 44

dermis of minipigs treated with nano TiO2 (Sadrieh et al., 2010 (Ref 199)). There is, 45 however, a consistent and large body of evidence from the submitted studies, and other 46

studies published in open literature (e.g. NANODERM, 2007; Nohynek et al., 2007), which 47

shows that nanoparticles do not penetrate deep enough to reach the viable epidermis or 48 dermis cells of healthy skin. In psoriatic skin, Pinheiro et al. (2007) showed that nano-TiO2 49

in a sunscreen formulation penetrated into deeper areas of the stratum corneum than in 50 healthy skin, but did not reach living cells in either psoriatic or healthy skin. Some in vitro 51

test systems, however, lack a stratum corneum layer, which can block penetration of TiO2 52 nanoparticles. Toxicological effects from such tests therefore need a careful consideration 53

since they may be difficult to extrapolate to the effects in vivo (Nohynek et al., 2007). 54 55

56

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A recent study by Bennett et al. (2012) investigated the penetration of TiO2 particles 1 through isolated pig skin sections and found a small fraction of the total dose in the skin 2

sections. The study found nanoparticles, or small clusters, in the interstitial spaces of the 3 porcine dermis after irradiation up to 500 µm depth, in comparison to the control skin 4

samples (tested under dark) where TiO2 was only found on the surface of the stratum 5 corneum. This study does raise questions over the possible disagglomeration of nanoparticle 6

clusters and enhanced penetration of TiO2 nanoparticles into skin under use conditions. The 7 study used TiO2 (anatase, non-coated) material, the type which is not recommended in this 8

opinion. Further studies will be needed on different crystalline forms and coated materials to 9 draw any conclusions on other TiO2 nanomaterials. 10

11

Contrary to the strong evidence suggesting a lack of penetration of TiO2 nanoparticles to 12 viable epidermis or dermis cells, there are a number of studies (in this submission and 13

published elsewhere), which indicate that nanoparticles can enter hair follicles. According to 14 SCCP opinion (2007) and NANODERM report (2007), adverse effects are not expected from 15

dermal exposure of healthy unflexed skin to photostable nano-TiO2 in sunscreens. However, 16 if photocatalytic nano-TiO2 is present in a sunscreen, it can potentially lead to generation of 17

reactive oxygen species (ROS) on exposure to UV light. 18 19

Most, if not all, studies provided in the submission were performed with nano TiO2 as 20

present in sunscreen formulations depicting consumer use. The studies were not directed 21 towards hazard identification using either a dose response approach or a worst case 22

scenario (overdosing situation). It is also of note that currently there are certain knowledge 23 gaps in relation to the possible dermal penetration of nano TiO2 on repeated or long term 24

use of cosmetic products, which may not only be used on flexed healthy skin but also on 25 skin that may have lesions or cuts. Studies provided in support of this submission have 26

shown that TiO2 nanoparticles do not penetrate the (simulated) sunburnt skin, whereas 27 such information on flexed or damaged skin is currently not available. 28

29

1.5.5 Repeated dose toxicity 30

31

1.5.5.1 Repeated Dose (30 days) oral toxicity 32

33

Exploratory subchronic oral study – Mice 30 day oral (gavage) 34 35

Guideline: No guideline 36 Species/strain: Mice/CD-1 37

Group size: 20 females per group 38

Test substance: TiO2 (Anatase, prepared from hydrolysis of Ti-tetrabutoxide, Primary 39 particle size 5 nm) 40

Batch: 41 Purity: 42

Vehicle: 43 Dose levels: 0, 62.5, 125 and 250 mg/kg bw/day 44

Dose volume: 45 Route: Oral 46

Administration: Intragastric administration every other day for 30 days 47

GLP: No 48 Study period: 2009 49

Reference: SI-II-Duan et al., 2010, (140) 50 51

Results 52

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Mice treated with doses ≥125 mg/kg bw/d showed body weight reduction, an increase in 1 coefficients of the liver and increased coefficients of the liver, kidney, spleen and thymus 2

and serious damage to liver function as shown by: 3 A decrease in interleukin-2 activity, white blood cells, red blood cells, haemoglobin, 4

mean corpuscular haemoglobin concentration, thrombocytes, reticulocytes, T 5 lymphocytes (CD3+, CD4+, CD8+), NK lymphocytes, B lymphocytes, and the ratio of 6

CD4 to CD8 of mice. 7

An increase in NO level, mean corpuscular volume, mean corpuscular haemoglobin, red 8

(cell) distribution width, platelets, hematocrit, mean platelet volume of mice. 9

Disruption of the liver function in terms of enhanced activities of alanine 10

aminotransferase, alkaline phosphatase, aspartate aminotransferase, lactate 11

dehydrogenase and cholinesterase, increase of total protein, and reduction of albumin to 12 globulin ratio, total bilirubin, triglycerides, and the total cholesterol levels. 13

No such effects were seen at low dose, and the NOAEL appears to be 62.5 mg/kg bw/d. 14 15

SCCS Comment 16 NOAEL derived from this study is 62.5 mg/kg bw/d. 17

1.5.5.2 Sub-chronic (90 days) toxicity (oral, dermal) 18

19

Subchronic oral toxicity – Rat 90 day oral (diet) 20

21 Guideline: No guideline 22

Species/strain: Rat/F344 23 Group size: 10 m, 10 f per group 24

Test substance: TiO2 (uncoated, Unitane®, Anatase), CAS No. 13463-67-7 25 Batch: 402110C46 26

Purity: 98% 27 Vehicle: 28

Dose levels: 6250, 12500, 25000, 50000, 100000 ppm 29


Administration: Diet 32 GLP: No 33

Study period: 1978 34 Reference: I-NCI, 1979 (22); DHS-NCI, 1979 (9) 35

36 Results 37

No deaths, no differences in body weight gains, no substance-related gross or microscopic 38

pathological finding, NOAEL: 100000 ppm. 39 40

SCCS Comment 41 No information has been provided on the particle size profile of the material tested in this 42

study. The study is therefore of little value in relation to the current assessment for nano-43 forms of TiO2. 44

45 Note 46

The two references provided (I-NCI, 1979 (22) and DHS-NCI, 1979 (9)) are in fact the 47

same. 48 49

50 Subchronic oral toxicity – Mouse 90 day oral (diet) 51


Species/strain: Mouse/B6C3Fi 54

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Group size: 10 m, 10 f per group 1 Test substance: TiO2 (uncoated, Unitane®, Anatase), CAS No. 13463-67-7 2

Batch: 402110C46 3 Purity: 98% 4

Vehicle: 5 Dose levels: 6250, 12500, 25000, 50000, 100000 ppm 6


Administration: Diet 9 GLP: No 10

Study period: 1978 11

Reference: I-NCI, 1979 (22); DHS-NCI, 1979 (9) 12 13

Results 14 No deaths, no differences in body weight gains, no substance-related gross or microscopic 15

pathological finding, NOAEL: 100000 ppm. 16 17

18 SCCS Comment 19

No information has been provided on the particle size profile of the material tested in this 20

study. The study is therefore of little value in relation to the current assessment for nano-21 forms of TiO2. 22

Note: Two references provided (I-NCI, 1979 (22); DHS-NCI, 1979 (9)) are in fact the same. 23 24

25 Exploratory subchronic oral study – Mice 60 day oral (gavage) 26


Species/strain: Mice/CD-1 29

Group size: 20 females per group 30 Test substance: TiO2 (Anatase, prepared from hydrolysis of Ti-tetrabutoxide, Primary 31

particle size 5 nm) 32 Batch: 33

Purity: 34 Vehicle: 35

Dose levels: 0, 5, 10, 50 mg/kg bw/d 36 Dose volume: 37

Route: Oral 38

Administration: Intragastric administration every day for 60 days 39 GLP: No 40

Study period: 2010 41 Reference: SI-II- Hu et al., 2010 (163) 42

43 Results 44

Potential effects on nervous system function, significant impairment of the behaviours of 45 spatial recognition memory. Indications for impaired neurofunction and behaviour at all 46

dose levels, indicated by: 47

Significantly altered levels of Ca, Mg, Na, K, Fe and Zn in brain 48

Inhibition of the activities of Na+/K+-ATPase, Ca2+-ATPase, Ca2+/Mg2+-ATPase, 49

acetylcholine esterase, and nitric oxide synthase; 50

Disturbed function of the central cholinergic system – significantly decreased levels of 51

monoamines neurotransmitters such as norepinephrine, dopamine and its metabolite 3, 52 4- dihydroxyphenylacetic acid, 5-hydroxytryptamine and its metabolite 5-53

hydroxyindoleacetic acid, 54

Increased levels of acetylcholine, glutamate, and nitric oxide. 55

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SCCS Comment 1 From the 60 day oral (gavage) study in mice, a LOAEL of 5 mg/kg bw/d may be derived. 2

3 4

1.5.5.3 Chronic (> 12 months) toxicity 5

6

No study provided 7 8

9 SCCS Comment on Repeated Dose Toxicity: 10

Two out of the 4 subchronic studies provided are of little value to the assessment of nano-11

forms of TiO2 because particle size distribution of the tested materials is not provided. The 12 other two studies used anatase nanomaterials. From the 60 day oral (gavage) study in 13

mice, a LOAEL of 5 mg/kg bw/d may be derived. 14 15

16

1.5.6 Mutagenicity / Genotoxicity 17

18 There are a number of issues in regard to in vitro testing of nanomaterials for mutagenicity. 19

Bacterial mutagenicity assays are considered to be less appropriate for the testing of 20

nanoparticles compared to mammalian cell systems due to the lack of endocytosis by 21 bacterial cells (EFSA, 2011). Therefore, for a negative outcome of such tests to be 22

acceptable, it is essential that contact of the test materials with bacterial DNA (i.e. 23 nanoparticle uptake by bacteria) is demonstrated. Furthermore, for testing of (conventional) 24

chemical substances, generally accepted positive controls are used for the various 25 Salmonella strains. The use of such chemical positive controls in testing nanomaterials 26

would not provide a proof for a negative response of the nanomaterial. Currently, there is 27 no accepted nanoparticle positive control that can demonstrate whether the assay is 28

suitable for the mutagenicity testing of insoluble/poorly soluble nanoparticles. 29

30 It is of note that the following studies have not been reviewed as part of this assessment 31

because they relate to test materials that are either not nanomaterials, or lack data on 32 material characterisation to establish whether they were relevant nanomaterials for this 33

assessment. 34 35

SI-Dunkel et al., 1985 (32); SI-Tennant et al. 1987 (33 (i, ii)); SI-Ivett et al., 1989 (35); 36 SIII-Lu et al., 1998 (56c), Nohynek, 1999 (56), PSMA statement, 1999 (66); SI-II Warheit 37

et al., 2007 (215); SIII-Lu et al., 1998 (56c), SI-Myhr, Caspary, 1991 (34); SI-Poole et al. 38

1986, (36); SI-Lemaire et al., 1982 (37); SI-II Msiska et al., 2010 (183); SI-Casto et al., 39 1979 (38); SI-Mikalsen et al., 1988 (39); SI-DiPole, Casto, 1979 (40); SI-Tripathy et al., 40

1990 (44); SI-Kitchin, Brown, 1989 (43); SI-II Pan et al., 2010 (189), SI-II DiVirglilio et 41 al., 2010 (139), Osman et at 2012 (188). 42

1.5.6.1 Mutagenicity / Genotoxicity in vitro 43

44

Bacterial gene mutation test 45 Guideline/method: OECD 471 (1997) 46

Test system: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and 47

Escherichia coli WP2uvrA. Tests were performed in absence or presence 48 of S9-mix 49

Replicates: Triplicate cultures in 2 independent experiments 50 Test items: T805 (coated, A/R, PSMA 1 type) 51

Batch: / 52 Solvent: Ethanol 53

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Concentrations: 8, 40, 200, 1000 and 5000 μg/plate in 1st experiment (range findings 1 experiment); 312.5, 625, 1250, 2500 and 5000 μg/plate in 2nd 2

experiment 3 Exposure: 48 h using the direct plate incorporation method 4

Negative control: yes (vehicle) 5 Positive control: ENNG for WP2uvrA, TA100 and TA1535; 9AA for TA1537 and 4NQO for 6

TA98; 2AA in all strains in experiments with S9-mix. 7 GLP: in compliance 8

Date of report: 19 June 1994 – 25 August 1994 9 Reference: Submission DHS (11), II(67) 10

11

The test substance was tested for mutagenicity in bacterial gene mutation assays with and 12 without metabolic activation (S9-mix prepared from Arochlor 1254 induced male Sprague 13

Dawley rat livers) using the direct plate incorporation method. Test concentrations were 14 based on the results of a preliminary toxicity study. The S. typhimurium strains TA98, 15

TA100, TA1535 and TA1537, and the E. coli strain WP2uvrA- were exposed for 48 h to the 16 test substance (suspended in ethanol) in concentrations ranging from 8 - 5000 μg/plate (1st 17

experiment) and 312.5 - 5000 μg/plate (2nd experiment). 18 19

Results 20

The test substance caused no visible growth reductions. Precipitation was observed at 21 concentrations of 625 μg/plate and above. All positive controls showed marked effects on 22

revertant colony numbers and the ethanol vehicle tested negative. Exposure to the test 23 substance did not result in biologically relevant increases in revertant colony numbers. 24

25 Conclusion 26

Under the experimental conditions used T805 was not mutagenic in this gene mutation tests 27 in bacteria. 28

29

SCCS Comment 30 See comments under 3.3.6 on the issues relating to the suitability of bacterial mutagenicity 31

assays for nanomaterials. 32 33

34 Bacterial gene mutation test 35

Guideline/method: OECD 471 (1983) 36 Test system: Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537. 37

Tests were performed in absence or presence of S9-mix 38

Replicates: Triplicate cultures in 2 independent experiments 39 Test items: T817 (coated, A/R, PSMA 1 type) 40

Batch: 04095 41 Solvent: Ethanol 42

Concentrations: 33.3, 100, 333.3, 1000, 2500 and 5000 μg/plate 43 Exposure: 48 h using the direct plate incorporation method 44

Negative control: vehicle 45 Positive control: NaN3 for TA100 and TA1535; 4-NOPD for TA1537 and TA98; 2AA in all 46

strains in experiments with S9-mix. 47

GLP: in compliance 48 Date of report: 1997 49

Reference: Submission DHS (12), II(67) 50 51

The test substance was tested for mutagenicity in a bacterial gene mutation test with and 52

without metabolic activation (S9-mix was prepared from phenobarbital/-naphtoflavone 53

induced male Wistar Rat livers). The S. typhimurium strains TA98, TA100, TA1535 and 54 TA1537 were exposed for 48 h to the test substance (suspended in ethanol) at 55

concentrations ranging from 33.3 to 5000 μg/plate. 56 57

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Results 1 Normal background growth was observed up to 5000 μg/plate. All positive controls showed 2

distinct increases in revertant colony numbers. Exposure to the test substance did not result 3 in biologically relevant increases in revertant colony numbers. 4

5 Conclusion 6

Under the experimental conditions used T817 was not mutagenic in this gene mutation tests 7 in bacteria. 8

9 SCCS Comment 10

The study is on T817 (coated, A/R, 95%, PSMA 1 type) which relates to Eusolex T in the 11

dossier. This study is relevant to the nanomaterial group (anatase). 12 See comments under 3.3.6 on the issues relating to the suitability of bacterial mutagenicity 13

assays for nanomaterials. 14 15

Bacterial Gene Mutation Test 16 Guideline/method: OECD 471 (1997) 17

Test system: Salmonella typhimurium strains T 98, T 100, T 102, T 1535 and TA1537, 18 in presence or absence of S9-mix 19

Replicates: Triplicate plates 20

Test items: T-LiteTM SF, pure rutile, primary particle size 10 x 50 nm, mean 21 agglomerates approximately 200 nm (d10: 90 nm, d90: 460 nm); 22

coating consisting of aluminium hydroxide and dimethicone/methicone 23 copolymer 24

T-LiteTM MAX, pure rutile, primary particle size 10 x 50 nm, mean 25 agglomerates approximately 200 nm (d10: 90 nm, d90: 460 nm); 26

coating consisting of dimethoxydiphenylsilane, triethoxycaprylylsilane 27 crosspolymer, hydrated silica and aluminium hydroxide 28

Batch: / 29

Solvent: DMSO (SPT), FCS (PIT) 30 Concentrations: 0, 20, 100, 500, 2500, 5000 μg/plate 31

Exposure: Standard plate test and or preincubation test 32 GLP: in compliance 33

Reference: Landsiedel et al., 2010 34 35

The test substances were tested for mutagenicity in the reverse mutation assay in bacteria 36 with and without metabolic activation. The S9 fraction was prepared from phenobarbital/β-37

naphthoflavone induced male Wistar rat liver. Both the standard plate test (SPT) and the 38

plate incorporation test (PIT) were used. The S/ typhimurium strains TA98, TA100, TA102, 39 TA1535 and TA1537 were exposed to the test substance (dissolved in DMSO (SPT) or fetal 40

calf serum (PIT)) at concentrations ranging from 20–5000 μg/plate. For control purposes, 41 DMSO) as negative control and the positive controls (NOPD, MNNG, AAC, MIT.C, 2-AA) were 42

also investigated. 43

Results 44

With the T-LiteTM SF a weak bacteriotoxicity was occasionally observed from 2500 μg/plate 45 onward in the presence of S9-mix only. With T-LiteTM MAX no bacteriotoxicity was noted. 46

Precipitation of the test substance was recorded from 100 μg/plate onward for T-LiteTM SF 47

and from 2500 μg/plate with T-LiteTM MAX. 48 The test substances did not induce a biologically relevant increase in revertant colony 49

numbers in the bacterial strains at any concentration tested in the presence or absence of 50 metabolic activation. 51

52 Conclusion 53

Under the experimental conditions used T-LiteTM SFand T-LiteTM MAX were not mutagenic in 54 this gene mutation tests in bacteria. 55

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SCCS Comment 1 The tested materials relate to S75-K (94% rutile, coated with aluminium hydroxide, 2

dimethicone/methicone copolymer). See comments under 3.3.6 on the issues relating to the 3 suitability of bacterial mutagenicity assays for nanomaterials. 4

5 6

Chromosome aberration test in mammalian cells 7 Guideline/method: OECD 473 (1997) 8

Test system: CHO cells. Tests were performed in absence or presence of S9-mix 9 Replicates: Duplicate cultures in 2 independent experiments 10

Test items: T805 (coated A/R, PSMA 1 type) 11


Concentrations: Experiment 1: 86.72, 209.7 and 800 μg/ml without S9 mix 14 167.8, 640 and 800 μg/ml with S9-mix 15

Experiment 2: 167.8, 512 and 800 μg/ml 16 Exposure: Experiment 1: 20 h treatment without S9 mix 17

3 h treatment and 17 h recovery with S9-mix 18 Experiment 2: 3 h treatment and 17 h recovery with S9-mix 19

Negative control: Vehicle 20

Positive control: NQO (without S9), CPA (with S9) 21 GLP: yes 22

Date of report: 17 November 1998 – 11 January 1999 23 Reference: Submission DHS (13), II(67) 24

25 The test substance was evaluated for potential cytogenetic effects in Chinese hamster ovary 26

(CHO) cells in the absence or presence of S9-mix. The S9 fraction was prepared from livers 27

of rats treated with Arochlor 1254 (experiment 1) or phenobarbital/-naphtoflavone 28

(experimen2t). Cytotoxicity was measured as a reduction in cell number compared to the 29 solvent control. In the absence of S9-mix only one experiment was performed. 4-30

nitroquilonine 1-oxide and cyclophosphamide were used as positive controls in the 31 experiments without and with S9-mix respectively. For each culture cells with structural 32

aberrations excluding gaps, and polyploidy, endoreduplication or hyperdiploidy were 33 categorized. 34

35

Results 36 The number of cells with structural aberrations in the negative control cultures were within 37

normal range. A biologically relevant increase in the number of cells with chromosome 38 aberrations was not observed due to exposure to T805 both without and with S9-mix. The 39

positive controls NQO and CPA induced statistically significant increases in the number of 40 cells with structural aberrations in the absence or presence of S9 mix respectively. 41

42 Conclusion 43

Under the experimental conditions used T805 was not genotoxic (clastogenic) in this 44

chromosome aberration test in mammalian cells. 45 46

SCCS Comment 47 The experiment in the absence of S9-mix was performed only once. 48

49 50

Chromosome aberration test in mammalian cells 51 Guideline/method: OECD 473 (1997) 52

Test system: CHO cells. Tests were performed in absence or presence of S9-mix 53

Replicates: Duplicate cultures in 2 independent experiments 54 Test items: T817 (coated A/R, PSMA 1 type) 55


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Concentrations: Experiment 1: 85.9, 640 and 800 μg/ml without S9-mix 1 167.8, 512 and 800 μg/ml with S9-mix 2

Experiment 2: 209.7, 512 and 800 μg/ml with S9-mix 3 Exposure: Experiment 1: 20 h treatment without S9 mix 4

3 h treatment and 17 h recovery with S9-mix 5 Experiment 2: 3 h treatment and 17 h recovery with S9-mix 6

Negative control: Vehicle 7 Positive control: NQO (without S9), CPA (with S9) 8

GLP: yes 9 Date of report: June 1999 10

Reference: Submission DHS (14), II(67) 11

12 The test substance was evaluated for potential cytogenetic effects in Chinese hamster ovary 13

(CHO) cells in the absence or presence of S9-mix. The S9-mix was prepared from livers of 14

rats treated with Arochlor 1254 (experiment 1) or phenobarbital/-naphtoflavone 15

(experiment 2). In the absence of S9-mix only one experiment was performed. Cytotoxicity 16

was measured as a reduction in cell number compared to the solvent control. 4-17

nitroquilonine 1-oxide and cyclophosphamide were used as positive controls in the 18 experiments without and with S9-mix respectively. For each culture cells with structural 19

aberrations excluding gaps, and polyploidy, endoreduplication or hyperdiploidy were 20 categorized. 21

22 Results 23

The number of cells with structural aberrations in the negative control cultures was within 24 normal range. In the experiment without S9-mix, a slight but not statistically significant 25

increase in the number of cells with chromosomal aberrations was observed. In the 26

experiments with S9-mix no biologically relevant increase in the number of cells with 27 chromosomal aberrations was observed. The positive controls NQO and CPA induced 28

statistically significant increases in the number of cells with structural aberrations in the 29 absence or presence of S9-mix, respectively. 30

31 Conclusion 32

Under the experimental conditions used T805 was not genotoxic (clastogenic) in this 33 chromosome aberration test in mammalian cells. 34

35

SCCS Comment 36 The experiment in the absence of S9 mix was performed only once. A tendency of an 37

increasing number of cells with structural aberrations was noted in the experiment without 38 S9-mix. 39

40 41

In vitro micronucleus test in human epidermal cells 42 Guideline/method: According to an generally accepted published protocol 43

Test system: Human epidermal cell line, A431 44

Replicates: 3 independent experiments 45 Test item: TiO2 NP (Anatase, 99.7%), commercial 46

Batch: / 47 Solvent: DMEM with 10% FBS 48

Concentrations: 0.008, 0.08, 0.8, 8, 80 µg/ml 49 Exposure: 6 h treatment without S9-mix, harvest time 24 h after the start of 50

treatment 51 Negative control: Vehicle 52

Positive control: Ethyl methanesulfonate (6 mM) 53

GLP: Not in compliance 54 Published: Shukla et al., 2011 55

Reference: Submission SI-II, (205) 56 57

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The cytokinesis-block micronucleus (CBMN) assay was carried out to determine the 1 potential genotoxicity of TiO2 NP in the human epidermal cell line A431. The cells were 2

treated for 6 h with different concentrations of TiO2 NP (0, 0.008, 0.08, 0.8, 8, and 80 3 µg/ml). Ethyl methanesulfonate was used as positive control. After the 6 h exposure, the 4

NPs were removed by washing with medium and cells were grown for additional 18 h in 5 fresh DMEM medium containing Cytochalasin-B (3 µg/ml medium). Cytospin preparations 6

were examined for the presence of micronuclei in binucleate cells. From each concentration 7 2000 binucleate cells were scored; the cytokinesis block proliferation index (CBPI) was 8

calculated from 500 cells/concentration as recommended in OECD Guideline 487. 9 Transmission electron microscopy (TEM) was used to evaluate uptake of the TiO2 NP into 10

the cells. 11

12 Results 13

CBPI was not significantly different from the control treatments. TEM analysis showed that 14 NPs were taken up by the cells. The NPs were found to be distributed mostly in cytoplasm, 15

some NP were also localised in the nucleus. A statistically significant induction in the 16 number of cells with micronuclei was observed after 6 h exposure to TiO2 NP. 17

The particles were also found to induce oxidative stress in the cells indicated by a significant 18 depletion of glutathione, induction of lipid peroxidation and reactive oxygen species 19

generation. 20

21 Conclusion 22

Under the experimental conditions used TiO2 NPs induced an increase in the number of cells 23 with micronuclei and, consequently, TiO2 NPs is genotoxic (clastogenic and/or aneugenic) in 24

the human epidermal cell line A431. 25 26

27 Fpg modified Comet assay in human epidermal cells 28

Guideline/method: According to an generally accepted published protocol 29

Test system: Human epidermal cell line A431 30 Replicates: 2 cultures 31

Test item: TiO2 NP (Anatase, 99.7%), commercial 32 Batch: / 33

Solvent: DMEM with 10% FBS 34 Concentrations: 0.008, 0.08, 0.8, 8, 80 µg/ml 35

Exposure: 6 h treatment 36 Negative control: Vehicle 37

Positive control: 25 µM hydrogen peroxide 38

GLP: Not in compliance 39 Published: Shukla et al., 2011 40

Reference: Submission SI-II, (205) 41 42

TiO2 NP was assayed for DNA damage in the human epidermal cell line A431 with the Comet 43 assay. The cells were treated for 6 hours with TiO2 NP in a concentration range up to 80 44

µg/ml. DNA damage was evaluated by formamidopyrimidine DNA glycosylase (fpg) modified 45 Comet assay. The fpg allows for detection of oxidative DNA base damage lesions, in 46

particular, 8-OH guanine. Hydrogen peroxide was included as a positive control and 47

cytotoxicity was evaluated by MTT and NRU assay. 48 49

Results 50 The TiO2 NP caused a significant concentration-dependent induction of DNA damage. Effects 51

were statistically significant at the two highest testing concentrations. These concentrations 52 were not cytotoxic after 6 or 24 h treatment in the MTT or NRU assay. Significant 53

cytotoxicity for both concentrations was found in these assays after 48 h treatment. Uptake 54 of NP into the A431 cells was shown by TEM analysis. Particles were observed mostly in the 55

cytoplasm, but occasionally also in the nucleus. Oxidative stress in the cells was indicated 56

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from the significant depletion of glutathione, induction of lipid peroxidation and reactive 1 oxygen species generation. 2

3 Conclusion 4

Under the experimental conditions used the results of the study indicate that TiO2 NPs 5 possess DNA damaging potential in human epidermal cells. 6

7 8

Comet assay in human lymphocytes 9

Guideline/method: According to an generally accepted published protocol for the alkaline 10

Comet assay 11

Test system: Human lymphocytes 12 Replicates: triplicate culture in 2 independent experiments 13

Test items: TiO2 NP commercial, declared size of 100 nm and surface area of 14.0 14 m2/g 15

Batch: / 16 Solvent: RPMI-1640 17

Concentrations: 0, 0.25, 0.50, 0.75, 1, 1.25, 1.50, 1.75, 2 mM 18 Exposure: 3 hour treatment 19

GLP: Not in compliance 20

Published: Ghosh et al., 2010 21 Reference: Submission SI-II, (157) 22

23

The DNA damaging potential of TiO2 NP was evaluated using the Comet assay in human 24

lymphocytes obtained by venipuncture from peripheral blood of healthy volunteers. Cells 25 were isolated by gradient centrifugation using Histopaque and resuspended in RPMI-1640 26

culture medium. Cells were treated for 3 hours with the TiO2 NP at a concentration range of 27 0 to 2mM. The Comet assay was performed according to published methods. DNA damage 28

was reported as % tail DNA in treated lymphocytes. Slides were prepared in triplicates per 29

concentration and each experiment was repeated twice. Viability was determined by trypan 30 blue dye exclusion, MTT assay and WST-1 assay in the same concentration range as used 31

for the Comet assay. 32

Results 33

Trypan blue indicated viability above 80% at the highest treatment concentrations. MTT and 34 WST-1 assay showed increased toxicity, with an LC50 in the range of 1.0 to 1.25 mM. A 35

statistically significant increase in DNA damage was observed in lymphocytes treated with 36 the TiO2 NP at a concentration of 0.25 mM. No concentration dependent effect and no 37

statistically significant effects were found at any of the other testing concentrations. 38

39 Conclusion: 40

Under the experimental conditions used, the results of the study indicate that TiO2 NPs 41 possess DNA damaging potential in human epidermal cells. 42

43

SCCS Comment 44

The authors of the paper conclude that TiO2 NP were genotoxic to human lymphocytes. 45 They propose that the absence of a dose-dependent effect on DNA damage may be due to 46

the agglomeration behaviour of the nanoparticles. 47

SCCS concludes that in view of the absence of a dose-dependent effect, this study does not 48 provide evidence for the genotoxicity of TiO2 in human lymphocytes. 49

50 51

In vitro mammalian cell gene mutation test 52 53

Guideline/method: / 54 Test system: gpt delta transgenic mouse primary embryo fibroblasts 55

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Replicates: / 1 Test items: TiO2 NP anatase (5 nm, 114m2/g), Sigma Aldrich, 2

TiO2 NP anatase (40 nm, 38 m2/g), Inframat Advanced Materials LLC 3 Fine TiO2 (325 mesh, 8.9m2/g), Sigma-Aldrich 4

Batch: / 5 Solvent: / 6

Concentrations: 0, 0.1, 1, 10 and 30 µg/ml 7 Exposure: 24 h treatment 8

Solvent: Distilled water (sonicated and then further diluted in culture medium) 9 GLP: not in compliance 10

Reference: Xu et al., 2009 11

12

Mutant frequencies in red/gam loci by Spi- detection by two nano-sized and one fine TiO2 13

materials were evaluated in gpt delta transgenic mouse primary embryo fibroblasts. The 14

samples were suspended in distilled water, subsequently sonicated for 30 min, sonicated on 15 ice, and diluted in medium before addition to the cells. S9-mix was not included in the 16

assay. 17 18

Results 19 Concurrent cytotoxicity of the samples was evaluated by the MTT assay and uptake of the 20

NP in the cells was assessed by flow-cytometry. Increased mutants frequencies were 21 observed with both the nanosize TiO2 samples, but not with the fine TiO2, demonstrating 22

that these nanomaterials can cause kilo-base pair deletion mutations. These effects could be 23

abrogated by co-treatment of the endocytosis inhibitor (lipid raft/caveolae disrupting agent) 24 Nystatin, the nitric oxide synthase inhibitor, NG-methyl-L-arginine (L-NMMA) and the 25

cyclooxygenase-2 activity inhibitor NS-398. 26 27

Conclusions 28 TiO2 NP were taken up by the cells and induced kilo-base pair deletion mutations in a 29

transgenic mouse mutation system. 30 It was suggested that induction of [ONOO]-, triggered by the signalling events associated 31

with the transporting of nanoparticles into the cells, rather than the chemical 32

composition/surface area combination of the nanoparticles may be a critical event for the 33 observed genotoxicity. 34

35 SCCS Comment 36

Translocation and contact of the test items with the fibroblast DNA has not been 37 demonstrated in the tests. The effects of the applied inhibitors suggest an indirect effect 38

mediated by TiO2 triggered formation of reactive oxidants. Uptake cannot be verified by 39 flow-cytometry on the basis of side-scattering, as particles may merely be adhered to the 40

cell membranes. 41

42 43

In vitro micronucleus test in mammalian cells 44 Guideline/method: Draft OECD 487 45

Test system: V79 cells 46 Replicates: Quadruplicate cultures 47

Test item: T-LiteTM SF, pure rutile, primary particle size 10 x 50 nm, mean 48 agglomerates approximately 200 nm (d10: 90nm, d90: 460 nm); 49

coating consisting of aluminium hydroxide and dimethicone/methicone 50

copolymere 51 Batch: / 52

Solvent: FCS 53 Concentrations: 75, 150 and 300 µg/ml with 4 h exposure 54

18.8, 37.5 and 75 µg/ml with 24 h exposure 55 Exposure: 4 h treatment and harvest 24 h after start of the treatment 56

24 h treatment and harvest immediately after the end of treatment 57

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Positive control: Ethyl methanesulfonate 500 µg/ml 1 GLP: not in compliance 2

Reference: Landsiedel et al., 2010 3 4

Micronucleus formation was evaluated in V79 cells after treatment with TiO2 rutile NP coated 5 with aluminium hydroxide and dimethicone/methicone copolymere (T-LiteTM SF rutile). The 6

study was performed without S9 mix which was considered scientifically justified because of 7 the nanoparticulate nature of the material. Cells were either treated for 4 hours with 75, 8

150 and 300 µg/ml, followed by 24 h recovery, or for 24 h at concentrations of 18.8, 37.5 9 and 75 µg/ml without recovery. The concentrations were selected on the basis of pilot 10

experiment on cytotoxicity. Ethyl methanesulfonate was used as positive control. 11

12 Results 13

The occurrence of precipitation at higher concentrations influenced the toxicity assessment. 14 Concurrent evaluation of cytotoxicity by analysis of proliferation index (PI) demonstrated 15

the absence of cytotoxicity up to highest scorable concentrations. A biologically relevant 16 increase in the number of cells with micronuclei was not observed after exposure to T-LiteTM 17

SF. 18 19

Conclusions 20

Under the experimental conditions used T-LiteTM SF did not induce an increase in the 21 number of cells with micronuclei and, consequently, T-LiteTM SF is not genotoxic (clastogenic 22

and/or aneugenic) in V79 cells. 23 24

SCCS Comment 25 The test material relates to S75-K (94% rutile, coated with aluminium hydroxide and 26

dimethicone/methicone copolymer). Translocation and contact of the test material with the 27 V79 cells and its possible translocation into the nucleus and interaction with DNA has not 28

been demonstrated. 29

30 31

Alkaline Comet assay in mammalian lung cells 32 Test system: A549 human lung carcinoma cells 33

Replicates: Triplicate cultures 34 Test items: TiO2 synthesized by laser pyrolysis (spherical, 12 nm, 92 m2/g, 95% 35

anatase, PZC (point of zero charge) = 6.4) 36 TiO2 synthesized by laser pyrolysis (spherical, 21 nm, 73 m2/g, 90% 37

rutile) 38

TiO2-A25 AEROXIDE-P25 (spherical, 24 nm, 46 m2/g, 86% anatase, PZC 39 = 7.0) uncoated 40

TiO2 ref. 637262 from Sigma-Aldrich (Elongated, L: 68 nm, d: 9nm, 118 41 m2/g, 100% rutile) 42

TiO2 ref. T8141 from Sigma-Aldrich (spherical, 142 nm, 10 m2/g, 100% 43 anatase, PZC = 5.2) 44

Batch: / 45 Solvent: Ultrapure sterile water (pH5.5), suspended at 10 mg/ml, further diluted 46

in cell culture medium 47

Concentrations: 0 and 100 µg/ml 48 Exposure: 4 h, 24h and 48 h after start of the exposure 49

GLP: not in compliance 50 Reference: Jugan et al., 2011 51

52

DNA damage of five different types of TiO2 particles (which included AEROXIDE-P25) was 53 evaluated by alkaline Comet assay in A549 cells. No S9-mix was added to the test system. 54

Cells were treated with one concentration (100 µg/ml) for 4 h, 24 h and 48 h. Cytotoxicity 55 was evaluated by the MTT-assay. Electron microscopy was performed to evaluate uptake of 56

the test samples into the A549 cells after 4 h. 57

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1 Results 2

Electron microscopic evaluation demonstrated a rapid uptake of the various test materials 3 into the cytoplasm of the A549 cells. Samples were tested only at one concentration. 4

Cytotoxicity, evaluated by MTT-test, revealed that cell death was less than 25% for all 5 samples after 48 h of exposure. 6

DNA damage was significantly increased with all samples at 4 h, with three out of the five 7 samples at 24 h. After 48 h no significant increase was detected with the exception of one 8

sample (i.e. laser pyrolysis synthesized rutile, 21 nm). The uncoated sample (AEROXIDE-9 P25) caused a significant increase in DNA single strand breakage at treatment times of 4 10

and 24 h. For all smallest, including the uncoated sample cellular internalization and 11

accumulation into cytoplasm was reported. For one sample (i.e. 12 nm laser pyrolysis 12 synthesized), nanoparticles were found located in the nucleus. 13

It was concluded that several types of TiO2 can cause DNA single strand breaks. In parallel 14 investigations, they also showed capacity of TiO2 to cause formation of the oxidative DNA 15

damage lesion 8-OHdG as well as an inhibition of DNA (base excision) repair activity. In 16

contrast, they did not detect double strand breaks evaluated by H2AX 17

immunohistochemistry or clastogenic/aneugenic effects evaluated by micronucleus assay in 18 the same cells. 19

20 Conclusions 21

Under the experimental conditions used it was concluded that TiO2 nanoparticles have a 22 genotoxic potential in this alkaline Comet assay in mammalian lung cells. 23

24 25

Alkaline Comet assay in mammalian liver cells 26

Guideline/method: According to generally accepted and published protocols 27 Test system: Human hepatoblastoma cell line C3A 28

Replicates: Triplicate cultures 29 Test items: NM101 Anatase 9 nm (XRD), 4-8/50-100 nm; two different particle 30

types (TEM), 322 m2/g 31 NRCWE001 Rutile 10 nm (XRD), 80-400 (TEM), 99 m2/g 32

NRCWE002 Rutile 10 nm (XRD), 80-400 (TEM), 84 m2/g, negative 33 charged 34

NRCWE003 Rutile 10 nm (XRD), 80-400 (TEM), 84 m2/g, positive 35

charged 36 NRCWE004 Rutile approx. 100 nm (XRD), 1-4/10-100/100-200/1000-37

2000; five different types of particles (TEM) 38 Batch: / 39

Solvent: Distilled water with FCS 40 Concentrations: Three concentrations, i.e. LC20, ½ of LC20 and 2x LC20 41

Exposure: 4 h treatment 42 Positive controls: H2O2 43

GLP: not in compliance 44

Reference: Kermanizadeh et al., 2012 45 46

DNA damage in human hepatoblastoma C3A cell line was evaluated by the alkaline Comet 47 assay (evaluated as % tail DNA), with inclusion of fpg enzyme to detect oxidative DNA 48

damage. A total of five different types of TiO2 were tested at a concentration that caused 49 20% viability loss (LC20), as well as twice or half of this concentration. The toxicity was 50

evaluated by WST-1 assay (24 h treatment), the treatment time for the Comet assay was 4 51 hours. S9 mix was not included in the assays. 52

53

Results 54 Biologically relevant and small but statistically significant increases in DNA damage were 55

found with several of the samples. The most pronounced effects were seen with NM101 and 56 RWCE001. No biologically relevant increase in DNA damage was observed with the 57

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negatively charged RCWE003. In view of the observed effects in the presence of fpg (as well 1 as based on further analysis of oxidative stress markers in the study), the authors suggest 2

that the DNA damage effects are mediated by reactive oxygen species (ROS). 3 4

Conclusions 5 Under the experimental conditions used, it was concluded that short term exposure of liver 6

cells to some TiO2 particles caused small but significant increases in DNA damage. 7 8

SCCS Comments 9 Translocation and contact of the test material with the hepatoblastoma cells and its' possible 10

translocation into the nucleus and interaction with DNA have not been demonstrated. Some 11

of the effects are minor but are concentration dependent, this might become significant at a 12 certain exposure level. 13

14 Further mutagenicity/genotoxicity in vitro studies (open literature): 15

The in vitro mutagenicity genotoxicity studies on TiO2 nanomaterials have been recently 16 reviewed by Magdolenova et al. (2013). In many of these studies, particle size (and 17

chemistry) is not, or poorly specified in the publications. As such, these studies do not allow 18 for evaluation of the potential effects of the nanosize aspect of the potential genotoxicity of 19

TiO2 (Le Boeuf et al., 1996; Endo-Capron et al., 1993; Pelin et al., 1995; Miller et al., 1995; 20

Lu et al., 1998; Kamp et al., 1995; Dunford et al., 1997; Wamer et al., 1997). In several 21 studies only fine TiO2 was used (e.g. Driscoll et al., 1997; Van Maanen et al., 1999, in both 22

these studies TiO2 anatase 180nm with a BET value of 8.8 m2/g was used; Notably 23 however, one may argue that this sample contains a particle distribution “tail” in the 24

nanosize range). 25

Nagakawa et al. (1997) tested four TiO2 samples, i.e. 21 nm and 255 nm anatase and 255 26

nm and 420 nm rutile for DNA strand breaks by alkaline Comet assay in the mouse 27 lymphoma cell clone L5178Y/tk+/-. In the presence of UV/light all samples showed enhanced 28

DNA strand breaks at concentrations which also elicited cell death. Without irradiation only 29

the 255 nm anatase showed enhanced strand breakage. The 21 nm anatase sample was 30 also evaluated for the induction of chromosomal aberrations in the Chinese Hamster cell line 31

CHL/IU, for mutagenicity in the Salmonella typhimurium strains TA100, TA98 and TA102, 32 and colony formation in the L5178Y/tk+/- cells. Chromosomal aberrations (mainly polyploidy, 33

chromatid breaks and chromatid exchanges) were found only in the presence of UV/visible 34 light, and occurred at cyotoxic concentrations. In the absence of light the 21 nm anatase did 35

not elicit chromosomal aberrations in contrast to the positive control (ofloxacin). 36 Irrespective of UV/light irradiation, the 21 nm anatase failed to enhance the frequencies of 37

revertant Salmonella colonies or mutant L5178Y colonies, in contrast to the positive control 38

methyl methanesulfonate (MMS). 39 40

Linnainmaa et al. (1997) investigated micronucleus formation in rat liver epithelial cells 41 after treatment with various TiO2 samples in the presence or absence of UV light. Mitomycin 42

C was used as positive control. TiO2 samples were a 170 nm and a 20 nm anatase sample, 43 and a 20 nm coated rutile sample. The coated sample was prepared with aluminium 44

hydroxide and stearic acid. The sample was ethanol washed to remove the stearic acid 45 before treatment of the cells. In contrast to the positive control, none of the samples 46

induced an increase in cells with micronuclei. 47

48 Rahman et al. (2002) studied micronucleus formation in SHE fibroblasts after treatment 49

with fine TiO2 (>200nm) and nanosize TiO2 (20nm). Apart from size, no further details of 50 the samples were provided. Increased micronuclei were found only with the ultrafine TiO2. 51

The authors reported (but did not show in the manuscript) that further kinetochore-staining 52 experiments revealed indications for chromosomal non-disjunction during mitosis. The 53

nanosize TiO2 also elicited apoptosis shown by DNA fragmentation analysis and the 54 appearance of apoptotic bodies (transmission electron microscopy evaluation). 55

56

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Gurr et al. (2005) tested a variety of TiO2 samples for micronucleus formation as well as the 1 induction of oxidative DNA damage using the Fpg-modified Comet assay in BEAS-2B human 2

bronchial epithelial cells. The samples used were four different anatase samples, with 3 respective sizes of 10, 20, 200 and >200 nm, and one rutile sample with the size of 200 4

nm. Micronucleus induction was found with the 10 and 200 nm anatase sample, but not 5 with the >200 nm anatase and the 200 nm rutile samples. For the 20 nm anatase sample 6

no data were provided. Enhanced oxidative DNA damage (fpg-Comet assay) was observed 7 with the 10 and 20 nm anatase samples and with the 200 nm rutile. All other samples were 8

negative. Finally, the authors showed that a 1:1 mixture of 200 nm anatase and 200 nm 9 rutile caused stronger oxidative DNA damage than the 200 nm anatase or 200 nm rutile 10

alone. 11

12 Bhattacharya et al. (2009) investigated the genotoxicity of anatase TiO2 in BEAS-2B human 13

bronchial epithelial cells and IMR-90 human lung fibroblasts. The TiO2 nanoparticles caused 14 induction of the oxidative DNA adduct 8-OHdG in IMR-90 cells (measured by an ELISA 15

method), but did not cause increased strand breaks (measured by Comet assay) in the IMR-16 90 and BEAS-2B cells. Electron microscopy demonstrated that both particles translocated 17

near to nucleus, but were not found inside the nucleus, mitochondria or ribosomes. 18 19

Falck et al. (2009) investigated the genotoxicity of three TiO2 samples in BEAS-2B human 20

bronchial epithelial cells by the alkaline Comet assay and the micronucleus test. The 21 samples were a nanosize rutile sample coated with <5 SiO2 (10x40nm needle shaped, BET 22

132 m2/g), a fine rutile sample (<5 µm, 2 m2/g), and a nanosize anatase sample (<25 nm, 23 222 m2/g). Hydrogen peroxide and mitomycin-C were used as respective positive controls. 24

All samples showed mild but significant DNA damaging effects. The effects of the nanosize 25 rutile were much weaker than those of the nanosize anatase and fine rutile sample. The 26

nanosize anatase, in contrast to both other samples, also caused increased micronuclei. For 27 the observed DNA damaging and micronucleus effects mostly no clear dose-dependency 28

could be observed. It was also reported that the micronucleus scoring was difficult due to 29

the presence of the particles during microscopy. 30 31

Magdolenova et al. (2012a) showed in human TK6, EUE and Cos-1 cells that genotoxicity of 32 TiO2 (DNA damage and oxidised DNA lesions) measured by the Comet assay (with and 33

without fpg) depends on the stock dispersion protocol. The same TiO2 (Aeroxide P25, 34 primary particle size 21 nm, mixture of anatase /rutile), but prepared with different stock 35

dispersion protocol, following further with the same media and exposure conditions resulted 36 in differed state of agglomeration and gave different results. Larger agglomerates gave 37

positive results. Thus differences in stock dispersion preparation could explain contradictory 38

results published on the same nanoparticles. Magdolenova et al. (2012b) studied the 39 possible interference of TiO2 and other nanoparticles with the fpg enzyme in the Comet 40

assay but did not find this to cause any artefacts. 41 42

1.5.6.2 Mutagenicity/Genotoxicity in vivo 43

44

Open literature studies 45 46

Micronuclei in peripheral blood erythrocytes after oral uptake 47

Guideline/method: / 48 Species/strain: C57Bl/6Jpun/pun mice. 49

Group size: 5 mice/treatment group 50 Test substance: Aeroxide P25, Degussa/Evonik, primary particle size 21 nm, BET surface 51

area 50 m2/g, DLS in water: 21-1446 nm) 52 Batch: / 53

Vehicle: water 54 Dose levels: 0, 50, 100, 250, and 500 mg/kg bw (estimated dose) 55

Treatment: / 56

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GLP: not in compliance 1 Reference: Trouiller et al., 2009 2

3 Methods 4

C57Bl/6Jpun/pun mice, containing naturally occurring 70-kb internal duplication in the pink-5 eyed dilution (p) gene, were exposed via drinking water to the TiO2 NP. The suspensions 6

were ultrasonicated for 15 min before providing to animals. Water (with/without the NP) 7 was provided ad libitum during 5 days. Peripheral blood was collected and erythrocytes 8

were evaluated for the presence of micronuclei. The estimated exposures were 0, 50, 100, 9 250 and 500 mg/kg bw. The doses were estimated on the basis of estimated drinking water 10

consumption (set at 5 ml) and the average weight of the animals. The authors also 11

evaluated DNA damage, measured as 8-hydroxy-2’-deoxyguanosine in liver tissue by 12 HPLC/ECD analysis, and alkaline Comet assay in blood cells, but these were tested only at 13

one concentration (500 mg/kg bw). Moreover, DNA deletions were evaluated in the 14 offspring of pregnant C57Bl/6Jpun/pun mice treated for 10 days at 500 mg/kg bw/day, to 15

evaluate in utero effects. 16 17

Results 18 A biologically relevant increase in the number of peripheral blood erythrocytes after oral 19

administration of TiO2 NP was found in mice at the highest treatment dose only (500 20

mg/kg). This concentration also caused increased DNA strand breakage in white blood cells 21 (Comet assay), γ-H2AX foci in bone marrow cells, and 8-hydroxy-2′-deoxyguanosine 22

formation in liver cells. A 10-day exposure in pregnant mice also led to DNA deletions in 23 offspring. The TiO2 NP exposure also caused a mild but statistically significant increase in 24

systemic inflammation, as shown by qRT-PCR analysis of the mRNA expression of 25 proinflammatory genes (TNFalpha, IFNgamma, KC/IL-8) in peripheral blood. It was 26

concluded that oral TiO2 NP exposure causes genotoxicity in mice, possibly caused by a 27 secondary genotoxic mechanism associated with inflammation and/or oxidative stress. 28

29

Conclusions: 30 Under the experimental conditions used, Aeroxide P25 was genotoxic (clastogenic and/or 31

aneugenic) in human lymphocytes in vitro. 32 33

SCCS Comments 34 The test material relates to S75-G (anatase/rutile, not coated). However, the description of 35

the test material given in the paper suggests a different proportion of anatase and rutile 36 (75%:25%) than the proportion specified for S75-G. Data indicate genotoxic effects of TiO2 37

NP after oral exposure in mice in organs/tissues other than those that are in direct contact 38

via the exposure route (i.e. effects in blood, bone marrow, liver and foetuses). Insufficient 39 details have been provided in the article regarding methodology. This makes the findings of 40

the study of limited value to this risk assessment. 41 Further limitations of the study are: 42

- The work does not contain biokinetics, i.e. dosimetry cannot be accurately 43 determined. Actual intake of the NP is not measured, only indirect by calculation of 44

the amount of drinking water. Translocation of particles and accumulation in different 45 organs was also not determined. 46

- Potential local effects (histopathology, genotoxicity assays) in gastrointestinal tract 47

target cells are not provided, and thus do not allow for assessment of potential 48 effects on epithelial barrier integrity, inflammation and local mutagenicity. 49

- The effects were observed at a rather high dose (calculated cumulative oral dose of 50 500 mg/kg). The authors do not report whether these concentrations affect intestinal 51

physiology. The high surface burden of TiO2 NP in the G.I. tract may have significant 52 impact on the adsorption and transport of nutrients. 53

54 55

DNA double strand breakage in bone marrow cells after oral uptake 56

Guideline/method: According to published protocols 57

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Species/strain: C57Bl/6Jpun/pun mice. 1 Group size: 5 / treatment group 2

Test substance: Aeroxide P25, Degussa/Evonik, primary particle size 21nm, BET surface 3 area 50m2/g, DLS in water: 21-1446nm) 4

Batch: / 5 Vehicle: water 6

Dose levels: 0, 50, 100, 250, and 500 mg/kg bw (estimated dose) 7 Treatment: / 8

GLP: not in compliance 9 Reference: Trouiller et al., 2009 10

11

Methods 12 DNA double strand breaks were analysed by immunohistochemical detection of γ-H2AX foci 13

in C57Bl/6Jpun/pun mice exposed to TiO2 NP via drinking water. Bone marrow smears were 14 analysed after 5 exposure days for γ-H2AX foci, at estimated exposure of the mice to 0, 50, 15

100, 250, and 500 mg/kg bw TiO2 NP. 16 17

Results 18 Oral TiO2 NP caused increased γ-H2AX foci in a clear dose dependent manner, being 19

significant from the lowest dose (50 mg/kg bw) onwards. DNA double-strand break was 20

considered the most sensitive parameter among a variety of genotoxicity endpoints. It was 21 therefore concluded that oral TiO2 NP exposure causes DNA double strand breaks in bone 22

marrow of the mice and suggest that this may be caused by a secondary genotoxic 23 mechanism associated with inflammation and/or oxidative stress. 24

The TiO2 NP exposure also caused mild but significantly increased systemic inflammation, as 25 shown by qRT-PCR analysis of the mRNA expression of proinflammatory genes (TNFalpha , 26

IFNgamma, KC/IL-8) in peripheral blood. 27 28

Conclusions: 29

Under the experimental conditions used, Aeroxide P25 was genotoxic rats causing DNA 30 double strand breaks in bone marrow cells. 31

32 SCCS Comments 33

34 The test material relates to S75-G (anatase/rutile, not coated). Marked dose dependent 35

effects are observed, suggesting that the bone marrow may be a sensitive target for TiO2 36 nanoparticle (after oral uptake). Whether the nanoparticles actually reached this target is 37

not shown in the study. The effects were observed at high concentrations. Other limitations 38

of the study are: 39 - The work does not contain biokinetics, i.e. dosimetry cannot be accurately 40

determined. Actual intake of the NP is not measured, only indirect by calculation of 41 the amount of drinking water. Translocation of particles and accumulation in different 42

organs was also not determined. 43 - Potential local effects (histopathology, genotoxicity assays) in gastrointestinal tract 44

target cells are not provided, and thus do not allow for assessment potential effects 45 on epithelial barrier integrity, inflammation and local mutagenicity. 46

- The effects were observed at a rather high dose (calculated cumulative oral dose of 47

500 mg/kg bw). The authors have not reported whether these concentrations affect 48 intestinal physiology. The high surface burden of TiO2 NP in the G.I. tract may have 49

significant impact on the adsorption and transport of nutrients. 50 51

52 Comet assay in vivo in rat lungs (five day inhalation study) 53

Guideline/method: According to generally accepted and published protocols 54 Species/strain: Male Wistar Crl:W1 Han rats 55

Group size: 3 animals per group 56

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Test substance: T-LiteTM SF, pure rutile, primary particle size 10 x 50 nm, mean 1 agglomerates approximately 200 nm (d10: 90 nm, d90: 460 nm); 2

coating consisting of aluminium hydroxide and dimethicone/methicone 3 copolymere 4

Batch: / 5 Vehicle: / 6

Dose levels: 0 and 10 mg/m3/treatment/day 7 Treatment: 6 h/day for 5 consecutive days 8

GLP: not in compliance 9 Reference: Landsiedel et al., 2010 10

11

Rats were exposed by inhalation (head-nose exposure) for 6 hours on five consecutive days 12 to 0 or 10 mg/m3/treatment/day. DNA damage was evaluated by alkaline Comet assay in 13

the rat lung cells (isolated by in situ perfusion) from three animals per group. Viability of 14 the isolated cells was determined by trypan blue dye exclusion. Further parameters 15

evaluated included body weight, and bronchoalveolar lavage levels of LDH and ALP. 16 17

Results 18 The treated animals showed significantly increased LDH and ALP concentrations in BAL. 19

Average viability of the cells isolated for the Comet assay were 95% and 88.7% respectively 20

for air and TiO2 exposed animals. A biologically relevant increase in DNA damage was not 21 detected by the Comet assay. 22

23 Conclusion 24

Under the experimental conditions used it was concluded that T-LiteTM SF has a genotoxic 25 potential in this alkaline Comet assay in lung cells. 26

27 SCCS Comment 28

The test material relates to S75-K (94% rutile, coated with aluminium hydroxide and 29

dimethicone/methicone copolymere). The applied method is not yet validated, but 30 represents tissue that at least in part is directly exposed to the testing material. The 31

isolation procedure may have affected the background damage in the cells from the 32 animals. 33

34 35

Further mutagenicity/genotoxicity studies in vivo (open literature) 36 In specific animal studies no information is provided on the size of the particles used, or 37

only non-ultrafine samples were used for effects of nano-sized TiO2 (Shelby, 1993; Driscoll 38

et al., 1997). 39 40

Rehn et al. (2003) investigated oxidative DNA damage induction by two samples of TiO2 in 41 rat lungs after intratracheal instillation at the dosages of 0, 0.15, 0.3, 0.6 and 1.2 mg/kg 42

bw/day. The samples used were an untreated TiO2 and a trimethoxyoctylsilane-treated TiO2 43 sample, both approximately 20 nm. DQ12 crystalline silica was used as a positive control at 44

0.6 mg/kg. Oxidative damage induction was determined after 90 days by 45 immunohistochemical analysis of lung sections using an 8-oxoguanine antibody. Enhanced 46

oxidative DNA damage was not observed with the untreated or silanised TiO2 nanoparticles, 47

in contrast to the DQ12 crystalline silica. Analysis of markers of pulmonary inflammation 48 and toxicity at 3, 21, and 90 days indicated a strong progressing inflammation with DQ12 49

crystalline silica, whereas for both TiO2 samples only mild inflammatory effects were 50 noticed. Proliferation in lung tissue, as determined using Ki-67 staining, showed only minor 51

differences between control and TiO2 treated rats in contrast to DQ12 treated rats which 52 showed strong increase in % Ki-67 positive cells after 90 days. The contrasting observations 53

with regard to oxidative DNA damage induction and proliferation were considered to be due 54 to the marked contrasts in severity and persistence of pulmonary inflammation. 55

56

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Similar to these observations, Driscoll et al. (1997) have demonstrated the likely role of 1 pulmonary inflammation in driving mutagenesis in rat lungs after in vivo instillation of 2

different particles. These included a fine crystalline silica sample, a nano-sized carbon black 3 sample and a fine anatase TiO2 sample (180 nm median diameter, 8.8 m2/g). Mutagenicity 4

was studied by hprt-analysis of lung epithelial cells isolated from the lungs of female SPF 5 F334 Fischer rats, 15 months after intratracheal instillation of each of the particles at 10 6

mg/kg or 100 mg/kg. For the fine TiO2 sample, enhanced hprt-mutagenesis was observed 7 with 100 mg/kg, the dose which also elicited persistent lung inflammation, but not with the 8

10 mg/kg dose. Similar for the other particles used (carbon black, silica) in vivo 9 mutagenicity was only observed at doses that also caused persistent inflammation. The 10

inflammatory cells obtained by bronchoalveolar lavage from the particle-treated animals 11

were found to induce hprt-mutagenesis in a rat lung epithelia cell line in vitro. 12 13

14 SCCS Comments on Mutagenicity/Genotoxicity 15

From the studies discussed above, the potential to cause DNA damage has been clearly 16 demonstrated for some TiO2 nanomaterials. However, it is not clear how this relates to the 17

other nanomaterials presented in the submission. 18

19

1.5.7 Carcinogenicity 20

21 Two stage skin painting carcinogenicity studies 22

23 Study Design: Two stage mouse skin carcinogenicity (Initiator: DMBA) 24

Date of publication: Available online 30 November 2010. 25 Guideline/method: Two stage muse skin carcinogenicity test. Coated and uncoated 26

titanium dioxide nanoparticles were used as promoter with 7,12-27 dimethylbenz[a]anthracene (DMBA) as initiator. 28

Test system: CD1 (ICR) female mice. 29

Test substance: Industrial material-grades of coated (alumina and stearic acid) 30 titanium dioxide nanoparticles (CTDN, titanium dioxide content: 31

79.2%, spindle shape, long axis of 50–100 nm, short axis of 10–20 32 nm) and uncoated titanium dioxide nanoparticles (UCTDN, titanium 33

dioxide content: 96.0%, spindle shape, long axis of 50–100 nm, short 34 axis of 10–20 nm) from Ishihara Sangyo Kaisha, Ltd., Osaka, Japan. 35

Batch: No data 36 Concentrations: CTDN and UCTDN dispersed in Pentalan 408 (pentaerythrityl 37

tetraethylhexanoate) at concentrations of 5 mg/0.1 g, 10 mg/0.1 g 38

and 20 mg/0.1 g on ultra sonic cleaner. 39 Exposure: Twice weekly for 19 weeks 40

Solvent: Pentalan 408 (pentaerythrityl tetraethylhexanoate) 41 Negative control: Solvent 42

Positive Controls: 12-o-tetradecanoylphorbol 13-acetate (TPA) 43 GLP: No 44

Reference: Furukawa et al., 2011 45 46

This study was conducted to examine the promoter potential of coated and uncoated 47

titanium dioxide nanoparticles (CTDN and UCTDN) in a two-stage mouse skin carcinogenesis 48 model using 7 week old CD1 (ICR) female mice. Initiation treatment: 0.1 ml (0.1 mg) DMBA 49

or vehicle alone was applied to furclipped back skin one time, using a micropipetter with 50 disposable tips. Starting 1 week after the initiation treatment, aliquots of 5, 10 and 20 mg 51

of CTDN or UCTDN in 0.1–0.09 ml of Pentalan were applied using a disposable syringe and 52 glass spreader daily, or 0.2 ml (4 µg) of TPA were applied using a micropipetter twice 53

weekly for 19 weeks to the animals as post-initiation treatments. TPA was used as a 54

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positive control promoter. Pentalan 408 served as a vehicle control as well as negative 1 control. 2

3 No changes in survival rate, general condition and body weight related to the test materials 4

were observed. On macroscopic observation, 1–2 nodules/group on the skin were observed 5 in each group applied CTDN and UCTDN as well as the control group after DMBA initiation. 6

The nodules were histopathologically diagnosed as squamous cell hyperplasia, sebaceous 7 gland hyperplasia, squamous cell papilloma and keratoacanthoma. While in CTDN and 8

UCTDN experiments enlargement of the mandibular, pancreatic, lumbar region and 9 inguinofemoral lymph nodes, spleen and thymus was observed in mice given 5 and 10 mg 10

but not 20 mg, the lack of dose-dependence suggests no biological significance. 11

12 The study authors concluded that CTDN and UCTDN applied as promoter at doses of up to 13

20 mg/mouse did not increase the development of nodules. There were no significant 14 differences between the number of nodules in the negative control (no initiator) and the 15

experiments with TiO2 as promoter. In the positive control, DMBA as initiator and TPA as 16 promoter, 100% of the animals developed nodules. The authors concluded that titanium 17

dioxide nanoparticles do not possess promoter activity for mouse skin carcinogenesis. 18 19

SCCS Comment 20

The test material used in this study might be comparable to one type of materials included 21 in this dossier. It was a good experiment with a procedure that is generally accepted for 22

studying initiation and promoter activity. SCCS agree that under the experimental 23 conditions uncoated and alumina- and stearic acid- coated nano TiO2 do not show any 24

carcinogenic promoter activity. 25 26

Study Design: Two stage mouse skin carcinogenicity 27

Date of publication: Published 2012 28

Guideline/method: Two stage mouse skin carcinogenicity test. Coated and uncoated 29

titanium dioxide nanoparticles were used as promoter with 7,12-30 dimethylbenz[a]anthracene (DMBA) as initiator. 31

Test system: Female rasH2 mice and their wild-type counterparts CB6F1 mice and 32 CD1 mice. 33

Test substance: sTiO2 particles (rutile type, silicone coated, mean particulate diameter 34 35 nm) and ncTiO2 rutile type mean particulate diameter 20 nm) 35

were provided by Japan Cosmetics Association, Tokyo. 36 Batch: No data 37

Concentrations: 0, 50 and 100 mg/ml 38

Exposure: sTiO2 rasH2 mice 5 times a week for 8 weeks, CB6F1 mice 5 times a 39 week for 40 weeks. 40

ncTiO2 CD1 mice 2 times a week for 52 weeks 41 Solvent: sTiO2 silicon oil, ncTiO2 Pentalan 408 (pentaerythrityl 42

tetraethylhexanoate) 43 Negative control: Solvent 44

Positive Controls: sTiO2 no positive control, ncTiO2, 12-o-tetradecanoylphorbol 13-45 acetate (TPA) 46

GLP: No 47

Reference: Sagawa et al., 2012 48 49

TEM analysis showed that the shape of sTiO2 particles was generally round to oval while 50 ncTiO2 particles were more club shaped. The mean length of sTiO2 particles suspended in 51

silicone was 0.28±0.22 µm. The mean length of ncTiO2 particles suspended in Pentalan 408 52 was 4.97±0.50 µm. 53

54 sTiO2 nano particles 55

The skin on the backs of 7-week old female rasH2 mice and wild type CB6F1 mice was 56

shaved and the animals received a single topical application of 0.1 ml DMBA (0.2 mg). Two 57

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weeks later the animals were divided into 3 groups. Group 1 (control, only initiation than 1 vehicle) (15 mice of each strain) were painted with 0.2 ml silicone oil. Group 2 (15 mice of 2

each strain) were painted with 0.2 ml of 50 mg/ml sTiO2 suspended in silicone oil. Group 3 3 (15 mice of each strain) were painted with 0.2 ml of 100 mg/ml sTiO2 suspended in silicone 4

oil. Group 4 (control, no initiation)(15 mice of each strain) were painted with 0.2 ml of 100 5 mg/ml sTiO2 suspended in silicone oil without prior DMBA treatment. The mice were painted 6

5 times a week. The rasH2 mice were killed after 8 weeks and the wild-type CB6F1 mice 7 after 40 weeks. 8

9 rasH2 mice 10

The incidence of squamous cell papillomas was 100% in all groups (Group 1 – 3) of rasH2 11

mice treated with DMBA. No skin tumours were found in the group (Group 4) which was 12 only treated with sTiO2. The incidence of squamous cell carcinomas was 33% in Group 1 13

(only DMBA and silicone oil), 60% in Group 2 (DMBA + 10 mg TiO2), and 53% in Group 3 14 (DMBA + 20 mg TiO2). The difference in carcinomas was not significant. No difference was 15

found in the multiplicity of tumours. 16 17

CB6F1 mice 18 The incidence of squamous cell papillomas was 7% (1 mouse) in Group 1 (only DMBA and 19

silicone oil) and 13% (2 mice) in Group 2 and 3 (DMBA + 10 and 20 mg TiO2). No skin 20

tumours were found in the group (Group 4) which was only treated with sTiO2. The 21 incidence of squamous cell carcinomas was 7% (1 mouse in Group 1 (only DMBA and 22

silicone oil). No squamous cell carcinomas were found in any of the other groups. 23 24

ncTiO2 nano particles 25 The skin on the backs of 10-week old female CD1 mice was shaved and the animals 26

received a single topical application of 0.1 ml DMBA (0.2 mg). Two weeks later the animals 27 were divided into 4 groups. Group 1 (control, only initiation than vehicle) (16 mice) were 28

painted with 0.2 ml Pentalan 408. Group 2 (16 mice) were painted with 0.2 ml of 50 mg/ml 29

ncTiO2 suspended in Pentalan 408. Group 3 (15 mice) were painted with 0.2 ml of 100 30 mg/ml ncTiO2 suspended in Pentalan 408. Group 4 (positive control)(15 mice) were painted 31

with 0.2 ml of TPA 200 nmol/ml in acetone. Groups 1 – 3 were painted 2 times a week and 32 killed after 52 weeks. Group 4 was painted 4 times a week and killed after 40 weeks. 33

34 CD1 mice 35

The incidence of squamous cell papillomas was 19% (3 mice) in Group 1 (only DMBA and 36 silicone oil), 6% (1 mice) in Group 2 (DMBA + 10 mg TiO2) and 13% (2 mice) in Group 3 37

(DMBA + 20 mg TiO2). None of the mice in Groups 1 – 3 had developed squamous cell 38

carcinomas. In the positive control (DMBA + TPA), 87% (13 mice) had developed squamous 39 cell papillomas and 13% (2 mice) had squamous cell carcinomas. 40

41 SCCS Comment 42

The results indicate that ncTiO2 does not promote skin tumours in mice. With sTiO2 an 43 increase in the number of tumours was found among mice initiated with DMBA. The 44

increase was not significant and no conclusion can be drawn. 45 46

47

Two stage rat skin carcinogenicity 48

Study Design 49

Date of publication: Published 2012 50 Guideline/method: Two stage rat skin carcinogenicity test. Uncoated titanium dioxide 51

nanoparticles (ncTiO2) was used as promoter with 7,12-52 dimethylbenz[a]anthracene (DMBA) as initiator. 53

Test system: Male Hras128 rats and their wild-type counterparts SD rats. 54 Test substance: ncTiO2 rutile type mean particulate diameter 20 nm) were provided 55

by Japan Cosmetics Association, Tokyo. 56

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Batch: No data 1 Concentrations: 0, 100 and 200 mg/ml 2

Exposure: ncTiO2 Hras128 rats 2 times a week for 28 weeks and SD rats 2 times 3 a week for 40 weeks. 4

Solvent: Pentalan 408 (pentaerythrityl tetraethylhexanoate) 5 Negative control: Solvent 6

Positive Controls: None 7 GLP: No 8

Reference: Sagawa et al., 2012 9 10

TEM analysis showed that the shape ncTiO2 particles were clubbed shaped. The mean length 11

of the ncTiO2 particles suspended in Pentalan 408 was 4.97±0.50 µm. 12 13

ncTiO2 nano particles 14 The skin on the backs of 10-week old male Hras128 rats and wild type SD rats was shaved 15

and the animals received a single topical application of 0.5 ml DMBA (2.5 mg). Two weeks 16 later the animals were divided into 3 groups. Group 1 (control, only initiation than vehicle) 17

(17 Hras128 rats and 12 SD rats) was painted with 0.5 ml Pentalan 408. Group 2 (16 18 Hras128 rats and 12 SD rats) was painted with 0.5 ml (50 mg) ncTiO2 suspended in 19

Pentalan 408. Group 3 (17 Hras128 rats and 12 SD rats) was painted with 0.5 ml (100 mg) 20

ncTiO2 suspended in Pentalan 408. The rats were painted twice a week. The Hras128 rats 21 were killed after 28 weeks and the SD rats after painting for 40 weeks. 22

23 Hras128 rats 24

The incidence of squamous cell papillomas was 94% (16 rats) in Group 1 (only DMBA and 25 Pentalan 408), 88% (14 rats) in Group 2 (DMBA + 50 mg TiO2) and 94% (16 rats) in Group 26

3 (DMBA + 100 mg TiO2). None of the rats Groups 1 had developed squamous cell 27 carcinomas, while 13% (2 rats) in both Group 2 and Group 3 had developed squamous cell 28

carcinomas. 29

30 SD rats 31

The incidence of squamous cell papillomas was 25% (3 rats) in Group 1 (only DMBA and 32 Pentalan 408), 17% (2 rats) in Group 2 (DMBA + 50 mg TiO2) and 8% (1 rat) in Group 3 33

(DMBA + 100 mg TiO2). None of the rats in Groups 1 and 3 had developed squamous cell 34 carcinomas, while 17% (2 rats) in both Group 2 had developed squamous cell carcinomas. 35

36 SCCS Comment 37

This rat model is less developed than the mouse two-stage carcinogenicity model. Since 38

94% of the Hras rats treated with DMBA only developed tumours, the model is not adequate 39 and no conclusion can be drawn from the study. 40

41 Study Design: Two stage rat skin carcinogenicity (Initiator: UV-B irradiation) 42

Date of publication: 2011 43 Guideline/method: Exploratory Dermal UV-B initiated skin carcinogenesis promotion 44

study. 45 Test system: Rat/Sprague-Dawley (wild-type and transgenic Hras128). 10 weeks 46

old 47

Group size: 5 – 8 male and 5 – 8 female per group. 48 Test substance: TiO2 NP (uncoated, rutile type, R, PPS: 20 nm, Ishihara Sangyo 49

Kaisha, Japan) 50 Batch: No data 51

Concentrations: 0, 100 mg/ml per rat (0.5 ml on 9 cm²) 52 Route: Topical application 53

Exposure: 42 weeks with/without pre-irradiation with UV-B for 10 weeks 54 Source of UV-light: UV-B radiation unit, Dermaray 100, Eisai-Toshiba, Tokyo, Japan 55

Irradiation: UV-B: 800 mJ/cm²P, 2x/week for 10 weeks 56

Solvent: Pentalan 408 (pentaerythrityl tetraethylhexanoate) 57

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Negative control: Solvent 1 Positive Controls: None 2

GLP: No 3 Reference: Xu et al., 2011 4

5 The potential of TiO2 NPs (uncoated, R, PPS: 20 nm) to promote skin tumours after dermal 6

application after UV-B irradiation was studied in transgenic rats carrying the human c-Ha-7 ras proto-oncogene (Hras128 rats), known to be sensitive to chemically induced skin 8

carcinogenesis in males and mammary carcinogenesis in females, and their wild-type 9 counterparts. A total of 80 Hras128 rats and their wild-type siblings were investigated. 10

11

The size of TiO2 particles suspended in Pentalan 408 ranged from 10 nm to 300 μm (mean 12 size of 5.0 μm, median size of 4.6 μm) indicating that a large majority of the particles 13

formed aggregates in the Pentalan 408 suspension. 14 15

Group 1 (initiation and promotion) received ultraviolet B (UV-B) radiation (UV-B radiation 16 unit, Dermaray 100, Eisai-Toshiba, Tokyo, Japan) 2 times per week for 10 weeks at 800 17

mJ/cm2, on the shaved target skin, followed by painting with 0.5 ml of TiO2 suspended in 18 Pentalan 408 at 100 mg/ml on the shaved (9 cm²) area twice a week until sacrifice. Group 19

2 (negative control, initiation + vehicle) received UV-B radiation and painting with the 20

vehicle Pentalan 408 on the shaved area twice a week until sacrifice, and Group 3 (no 21 initiation, only TiO2 as promoter) received painting with 0.5 ml of TiO2 suspension as in 22

Group1 but without prior UV-B radiation. 23 24

Any grossly visible papilloma lesions were carefully examined every day. All the animals 25 were sacrificed at week 52 (after 42 weeks painting) except for the female Hras128 rats, 26

which were terminated at week 16 (after 6 weeks painting) due to early mammary tumour 27 development. The skin, brain, lung, liver, mammary gland, mesenterial lymph nodes, spleen 28

and kidney, were excised, fixed and processed for light microscopic examination. 29

30 In male Hras128 rats, papillomas on the back skin developed from week 32 and the 31

incidence of skin papillomas was 12.5% (1/8) in Groups 1 and Group 3. No skin tumours 32 were observed on the targeted back skin in female Hras128 rats or wild-type rats of either 33

sex. Eye lid squamous cell papillomas were found in wild type female rats exposed to UVB 34 (Groups 1 and 2) with incidences of 12.5% (1/8) and 14.3% (1/7). No statistically 35

significant inter-group differences in incidence, multiplicity or weight were found. Mammary 36 tumours (adenocarcinomas) were induced with high incidence in Hras128 rats of both 37

sexes. Wild-type female rats also had an increased incidence of mammary tumours but no 38

statistically significant inter-group differences in incidence, multiplicity or weight were 39 observed. 40

41 Conclusions by the authors 42

TiO2 particles were detected in the upper stratum corneum but not in the underlying skin 43 tissue layers. TiO2 did not induce or promote skin carcinogenesis in transgenic (Hras128) 44

and wild-type Sprague-Dawley rats under the conditions of this study. The data suggest 45 that TiO2 does not cause skin carcinogenesis, probably due to its inability to penetrate 46

through the epidermis and reach underlying skin structures. 47

48 SCCS Comment 49

This is not a generally accepted model for studying initiation and promotion of skin tumours. 50 Since no positive control was included it is not possible to make any conclusion with regard 51

to potential carcinogenic properties of TiO2 from the study. 52 53

Study Design: Intra-pulmonary spraying 54

Date of publication: Advance Access publication February 25, 2010. 55

Guideline/method: Two stage rat skin carcinogenicity test. Uncoated titanium dioxide 56

nanoparticles (ncTiO2) were used as promoter with DHPN as initiator. 57

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Test system: Female transgenic rats carrying the human c-Ha-ras gen (Hras128 1 rats) and female wild-type SD rats were obtained from CLEA Japan 2

Co., Ltd (Tokyo, Japan) 3 Test substance: ncTiO2 rutile type mean particulate diameter 20 nm) were provided 4

by Japan Cosmetics Association, Tokyo. 5 Batch: No data 6

Concentrations: TiO2 particles were suspended in saline at 250 µg/ml or 500 µg/ml. 7 Exposure: Initiation: 0.2% DHPN (N-nitrosobis(2-hydroxypropyl)amine), (Wako 8

Chemicals Co., Ltd Osaka, Japan) in the drinking water for 2 weeks. 9 Promotion: Two weeks after DHPN treatment, the rats were exposed 10

intratracheally every second week to TiO2 suspensions under 11

isoflurane anesthesia for a total of 7 times. The rats were killed 3 12 days after the last exposure. 13

Solvent: Saline 14 Negative control: Only DHPN in drinking water 15

Positive Controls: None 16 GLP: No 17

Reference: Xu et al., 2011 18 19

Female transgenic Hras128 rats and female wild-type SD rats were used in the study. TiO2 20

particles were suspended in saline at 250 µg/ml or 500 µg/ml. The TiO2 suspension was 21 intratracheally administered to animals under isoflurane anesthesia using a Microsprayer 22

(Series IA-1B Intratracheal Aerosolizer, Penn-Century, Philadelphia, PA) connected to a 1 ml 23 syringe; the nozzle of the sprayer was inserted into the trachea through the larynx and a 24

total of 0.5 ml suspension was sprayed into the lungs synchronizing with spontaneous 25 respiratory inhalation (IPS). 26

27 IPS-initiation–promotion protocol 28

Female Hras128 rats aged 6 weeks were given 0.2% DHPN, in the drinking water for 2 29

weeks. Two weeks later, the rats were divided into four groups. Group 1 (9 rats). DHPN 30 alone. Group 2 (10 rats). DHPN followed by 250 µg/ml TiO2. Group 3 (11 rats). DHPN 31

followed by 500 µg/ml TiO2. Group 4 (9 rats). 500 µg/ml TiO2 without DHPN initiation. 32 33

The TiO2 particle preparations were administered by IPS once every 2 weeks from the end 34 of week 4 to week 16 (a total of seven exposures). The total amount of TiO2 administered to 35

Groups 1, 2, 3 and 4 were 0, 0.875, 1.75 and 1.75 mg per rat, respectively. Three days 36 after the last treatment, animals were killed and the organs (brain, lung, liver, spleen, 37

kidney, mammary gland, ovaries, uterus and neck lymph nodes) were excised 38

39 TiO2 was distributed primarily to the lung, but minor amounts of TiO2 were also found in 40

other organs. Various sizes of TiO2 aggregates were observed in alveolar macrophages. The 41 TiO2-laden macrophages were evenly scattered throughout the lung alveoli. Of 452 particle 42

aggregates examined, 362 (80.1%) were nanosized, i.e.100 nm. Overall, the average size 43 was 84.9 nm and the median size was 44.4 nm. 44

45 The author concluded that TiO2 treatment significantly increased the multiplicity of DHPN-46

induced alveolar cell hyperplasias and adenomas in the lung. In the rats, which received 47

TiO2 treatment without prior DHPN treatment, alveolar proliferative lesions were not 48 observed although slight inflammatory lesions were observed. TiO2 aggregates were 49

localized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm. 50 51

In the mammary gland, TiO2 treatment significantly increased the multiplicity of 52 adenocarcinomas from about 3 tumours per rat in Group 1 to about 6 tumours per rat in 53

Group 2 and 3. The treatment did also tend to increase the weight of the mammary tumors 54 from about 6 g per tumour in Group 1 to about 12 – 15 g per tumour in Group 2 and 3 (only 55

shown in Figure with no Table). 56

57

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IPS 9 day protocol 1 Twenty female SD rats (wild-type counterpart of Hras128) aged 10 weeks were treated by 2

IPS with 0.5 ml suspension of 500 µg/ml TiO2 particles in saline five times over a 9 day 3 period. The total amount of TiO2 administered was 1.25 mg per rat. Six hours after the last 4

dose, animals were killed and the lungs and inguinal mammary glands were excised. Fatty 5 tissue surrounding the mammary gland was removed as much as possible. The left lungs 6

and inguinal mammary glands were used for biochemical analysis, and the right lungs were 7 fixed in 4% paraformaldehyde solution in PBS adjusted at pH 7.3 and processed for 8

histopathological examination and immunohistochemistry. 9 10

Morphologically, TiO2 particles were observed as yellowish, polygonal bodies in the 11

cytoplasm of cells. These cells are morphologically distinct from neutrophils and strongly 12 positive for CD68, indicating that the TiO2 engulfing cells were macrophages. TiO2 13

aggregates of various sizes were found in macrophages, and aggregates larger than a single 14 macrophage were surrounded by multiple macrophages. Of 2571 particle aggregates 15

examined, 1970 (76.6%) were <100 nm and five particles were >4000 nm in size. Overall, 16 the average size was 107.4 nm and the median size was 48.1 nm. 17

18 TiO2 treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide 19

dismutase activity and macrophage inflammatory protein 1a (MIP1a) expression in the lung 20

21 Comment by the authors 22

TiO2 treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide 23 dismutase activity, and macrophage inflammatory protein 1a (MIP1a) expression in the 24

lung. MIP1a, detected in the cytoplasm of TiO2-laden alveolar macrophages in vivo and in 25 the media of rat primary alveolar macrophages treated with TiO2 in vitro, enhanced 26

proliferation of human lung cancer cells. Furthermore, MIP1a, also detected in the sera and 27 mammary adenocarcinomas of TiO2-treated Hras128 rats, enhanced proliferation of rat 28

mammary carcinoma cells. These data indicate that secreted MIP1a from TiO2-laden 29

alveolar macrophages can cause cell proliferation in the alveoli and mammary gland and 30 suggest that TiO2 tumor promotion is mediated by MIP1a acting locally in the alveoli and 31

distantly in the mammary gland after transport via the circulation. 32 33

SCCS Comment 34 TiO2 treatment significantly increased the multiplicity of DHPN-induced alveolar cell 35

hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas. 36 Thus, non-coated TiO2 administered intratracheally had tumour promoter activity. 37

38

39 Oral carcinogenicity studies in non-nano TiO2 40

41 Oral study with F344 rats. Each groups consisted of 60 male and 60 female rats. The control 42

diet contained 1% corn oil, while experimental diets contained 1.0, 2.0, or 5.0% titanium 43 dioxide-coated mica and 1% corn oil. 44

45 The article states: “TiO2-coated mica is a nonfibrous, naturally occurring silicate, which, 46

when coated with TiO2 is used as a pearlescent pigment in plastics, industrial coatings, 47

simulated leather, and cosmetic preparations. Annual worldwide production of TiO2-coated 48 mica exceeds 1 million pounds and the potential for human exposure is great.” 49

50 The test material consisted of a 1:1 blend of two samples of titanium dioxide-coated mica. 51

The material was in the form of flat platelet with the longest dimension ranging from 10 to 52 35 µm. The final blend of test material contained 28% TiO2 and 72% mica. A purity of 53

100% was assumed for purposes of diet formulations. 54 55

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The rats (6 week old) received the TiO2 containing for up to 130 weeks. The study authors 1 stated that “there was no evidence that TiO2-coated mica produced either toxicologic or 2

carcinogenic effects at dietary concentrations as high as 5.0%. 3 Ref.: Bernard et al., 1990 4

5 Groups of 50 male and 50 female B6C3F1 mice, 5 weeks of age, were fed diets containing 6

0, 2.5 or 5% titanium dioxide (size unspecified; anatase; purity, ≥98%) daily for 103 7 weeks. Mice were killed at 109 weeks of age, at which time no significant difference in 8

survival was observed between treated and control males. In females, a dose-related trend 9 in decreased survival was noted. No significant differences in body weights or incidence of 10

tumours were observed between treated and control groups. 11

12 Groups of 50 male and 50 female Fischer rats, 9 weeks of age, were fed diets containing 0, 13

2.5 or 5% titanium dioxide (size unspecified; anatase; purity, ≥98%) daily for 103 weeks. 14 The rats were killed at 113 weeks of age, at which time no significant difference in survival 15

was observed between treated and control groups of either sex. No significant differences in 16 body weights or incidence of tumours were observed between treated and control groups. 17

Ref.: National Cancer Institute, 1979 18 19

SCCS Comment 20

From the studies, exposure to non-nano titanium dioxide via the oral route does not appear 21 to lead to carcinogenic effects. 22

23

1.5.8 Photo-carcinogenicity 24

25 Photo-carcinogenicity studies in non-nano TiO2 26

27 The ability of MTD (titanium dioxide, not further specified) and 2-EHMC (2-ethylhexyl-p-28

methoxycinnamate) to protect mice from the “promotion phase” of tumorigenesis was 29

studied. 30 31

The dorsal trunks of inbred female C3H/HeJ mice (10 – 12 weeks old) were shaved and the 32 relevant groups (15 mice) initiated with 10 nmol DMBA. Five days later UV-irradiation 33

and/or sunscreen treatment was commenced and this was continued for 32 weeks. The 34 mice were monitored for a further 14 weeks after cessation of irradiation. 35

36 The sunscreens were in oil-in-water emulsion and contained MTD (7.2%) or 2-EHMC (8%). 37

The MTD was a broad-spectrum-reflecting physical sunscreen with an SPF of 7, while the 2-38

EHMC was shown to be a UVB-absorbing sunscreen with an SPF of 4. The sunscreens or 39 base lotion (BL) were applied at least 10 min prior to UV exposure at approximately 2 40

mg/cm2. The integrated irradiance was 1.7 W/m2 for UVB and 34 W/m2 for UVA. 41 42

The mice were irradiated 5 days per week for 32 weeks, i.e. until 50% of the DMBA plus UV 43 irradiated groups had tumors. The average cumulative dose was 571 kJ/m2 for UVB and 44

11.4 mJ/m2. 45 46

The DMBA-initiation alone and DMBA-initiated sunscreen-treated groups did not develop 47

tumours. UV alone induced tumours in 46% of the mice at week 48. Initiation with DMBA 48 prior to UV irradiation enhanced tumour formation such that 87% had tumours at week 48. 49

Both MTD and 2-EHMC completely protected the mice from UV-induced tumour formation. 50 Ref.: Bestak and Haliday, 1996. 51

52 Groups of female inbred mice (hr/hr, strain Skh:HR-1) treated with an SPF 15 sunscreen 53

formulated with MT100T microfine titanium dioxide coated with aluminium stearate (not 54 further specified) were exposed daily to minimally skin reddening UV radiation over 12 55

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weeks. Throughout a 200 day observation period substantial protection was afforded from 1 the induction of skin cancer compared to unprotected controls. 2

3 Two groups of sunscreen protected mice were treated immediately following the radiation 4

regime with the tumour promoter croton oil. UV + croton oil induced tumours in 100% of 5 the mice. The mice protected by a sunscreen showed only 3.7% with tumours, which was 6

less than with treatment with croton oil alone. However, where sunscreen protected mice 7 were exposed to croton oil about 25% proved to have been initiated. 8

9 The authors concluded that the superfine titanium dioxide sunscreen provided a high level 10

of protection similar to that by conventional sunscreen formulations. 11

Ref.: Greenoak et al., 1993. 12 13

SCCS Comment 14 The studies above are of little value because size and specifications of the titanium dioxide 15

particles are unknown. 16 17

18 SCCS Comments on Carcinogenicity 19

Pigmentary and ultrafine titanium dioxide has been tested for carcinogenicity by oral 20

administration in mice and rats, by inhalation exposure in rats and female mice, by 21 intratracheal administration in hamsters and female rats and mice, by subcutaneous 22

injection in rats, and by intraperitoneal administration in male mice and female rats. 23 24

- According to the evaluation of titanium dioxide by IARC (2010), induction of lung 25

tumours was observed in two inhalation studies with rats while two inhalation studies in 26 rats and one in female mice gave negative results. 27

- Intratracheally instilled female rats showed an increased incidence of lung tumours 28

following treatment with two types of titanium dioxide. Tumour incidence was not 29 increased in intratracheally instilled hamsters and female mice. 30

- Oral, subcutaneous and intraperitoneal administration did not produce a significant 31

increase in the frequency of any type of tumour in mice or rats. 32

- IARC concluded that there is inadequate evidence in humans for the carcinogenicity of 33

titanium dioxide but sufficient evidence in experimental animals for the carcinogenicity 34

of titanium dioxide. Titanium dioxide was classified as a Group 2B carcinogen (Possibly 35 carcinogenic to humans). 36

- In their recent evaluation of TiO2 NIOSH has determined that ultrafine TiO2 with equal 37

nano-sized TiO2 is a potential occupational carcinogen and, that there is insufficient data 38 to classify fine TiO2 as potential occupational carcinogen after inhalation (NIOSH 2011). 39

- Nano titanium dioxide has been studied in 2 two-stage skin carcinogenicity studies with 40

mice, 2 two-stage skin carcinogenicity studies with rats, and one two-stage lung study 41 with rats. 42

- Both non-coated (ncTiO2) and coated titanium dioxide have been studied in the two-43

stage mouse skin carcinogenicity studies with CD1 mice and a transgenic mouse strain 44

(rasH2). In one well performed study with non-coated and alumina and stearic acid 45 coated titanium dioxide, no promoter activity was found (Furukawa et al., 2011). 46

Promoter activity was also not found for ncTiO2 in the other study (Sagawa et al., 2012). 47 However, it is difficult to draw a firm conclusion from this study with silica coated 48

titanium dioxide due to lack of positive controls and very high tumour activity in the 49 “initiated” mice. 50

- Non-coated titanium dioxide was studied in 2 two-stage rat skin carcinogenicity studies. 51

Although, no tumour promoter activity was observed, it is difficult to draw any 52

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conclusion since little experience with the model used is available and no positive 1 controls have been used in the studies. 2

- One two-stage rat lung carcinogenicity studyhas been carried out with non-coated 3

titanium dioxide. The rats were “initiated” by DHPN in the drinking water prior to intra-4 pulmonary spraying with ncTiO2. The experiment demonstrated promoter activity of 5

ncTiO2 (Xu et al., 2011). 6

Since TiO2 particles have shown carcinogenic activity and since nano ncTiO2 also showed 7

promoter activity after intra-pulmonary spraying, the use of nano TiO2 in sprayable 8 applications needs specific considerations. 9

10

1.5.9 Reproductive toxicity 11

12

In the submission, no studies have been provided with reproductive toxicity data relevant to 13 the nanomaterials under assessment. A review of reproductive and developmental toxicity 14

studies of manufactured nanomaterials (including TiO2) has been provided (Ema et al., 15 2010 - Reference 146). The two TiO2 materials referred to include a TiO2 material with 16

particle size <10µm (no further information), and a TiO2 nanomaterial with primary particle 17 size 25-70 nm (20–25m2/g surface area, anatase). Relevant studies in the review by Ema 18

et al. (2010) showed that: 19

- Pregnant BALB/c mice administered on gestational day 14 with <10 µm TiO2 20

suspended in phosphate-buffered saline at 50 µg/mouse by a single intranasal 21

insufflation had higher serum levels of cytokines, including interleukin-1β, tumor 22 necrosis factor-α, interleukin-6 and chemokine, 48 h after exposure compared with 23

nonpregnant mice. The offspring of the dams exposed to TiO2 showed increased 24 airway hyperresponsiveness, increased percentage of eosinophils, and pulmonary 25

inflammation. These findings showed that TiO2 caused acute cellular inflammation in 26 pregnant mice and increased allergic susceptibility in their pups. 27

- Pregnant Slc:ICR mice administered on gestational days 6, 9, 12 and 15 with TiO2 28 nanomaterial suspended in saline with 0.05% Tween 80 via subcutaneous injection 29

at 100µg/mouse/day caused changes in the expression of genes associated with 30

brain development, cell death, response to oxidative stress, and mitochondria in the 31 brain during the prenatal period, and genes associated with inflammation and 32

neurotransmitters in the later stages of the offsprings. 33

- In vitro exposure of testis-constituent cells (mouse Leydig cell line TM3) to nano-34

TiO2 showed uptake of the nanoparticles after incubation of cells at 30µg/mL for 35 48h, and a remarkable inhibition of viability and transient reduction in proliferation of 36

the cells at 100µg/mL after 24 h. 37

The article is, however, a review of exploratory studies, and as such is of a limited 38

usefulness to this assessment. 39

Other studies in open literature, including some of those reviewed by Ema et al. (2010) 40 have demonstrated the possibility of placental transport of different manufactured 41

nanomaterials in pregnant animals into the fetus, or found effects in the offspring. 42 Yamashita et al. (2011) reported on the presence of nano-TiO2 in fetuses after the 43

intravenous administration of nano-TiO2 in pregnant mice. Nano-TiO2 was detected by TEM 44 in the placenta, fetal liver and fetal brain, and induced a decrease in uterine weight and 45

higher fetal absorption. A limitation of the study was that relatively high doses (about 32 46 mg/kg body weight on gestation days 16 and 17) were used. In addition, the chemical 47

nature of the nanomaterials observed in the organs was not confirmed. For the silica 48

nanoparticles investigated in the same paper a size dependency of transplacental migration 49 was demonstrated as 70 nm nanoparticles did show placental transport while 300 nm and 50

1000 nm silica nanoparticles did not (Yamashita et al., 2011). 51

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After subcutaneous administration to dams (Slc:ICR mice) on gestation days 3, 7, 10 and 1 14 at 100µg/mouse/day, Takeda et al. (2009) observed TiO2 particle aggregates (identified 2

by Energy Dispersive X-ray Spectroscopy, EDS) in the testis of male offsprings at day 4 and 3 week 6 after birth. Also histopathological alterations were observed in the testis. In 4

addition, nano-TiO2 particles were demonstrated in the brain of offspring mice (Takeda et 5 al., 2009), suggesting that nano-TiO2 might have passed through undeveloped or 6

developing Blood Brain Barrier (BBB) in embryos of the young mice. However, since mice 7 were tested at 4 days or 6 weeks of age, it is not clear whether exposure to nano-TiO2 8

occurs in utero via the placenta or through milk. A previous study of the same research 9 group observed alterations in gene expression in the brain (Shimizu et al., 2009). The gene 10

expression alterations were already observed in 16 days old embryos. As only the mother 11

animals were exposed to nano-TiO2 it seems likely that the offspring received the Ti via the 12 mother either during pregnancy or in the weaning period via the milk (Takeda et al., 2009). 13

For some effects, like reduced pup weight and gene alterations, indirect mechanisms due to 14 effects on the pregnant animals themselves could not be excluded. 15

After inhalation exposure to nano-TiO2 during gestation days 8-18 moderate behavioural 16 effects were observed in the offspring (Hougaard et al., 2010). Time to first litter was 17

prolonged after mating the exposed male offspring to unexposed mice but did not reach 18 statistical significance. For females there was no difference. After inhalation of a surface 19

coated nano-TiO2 by pregnant mice, no effects were seen on DNA damage in 20

bronchoalveolar lavage fluid (BALF) cells and liver cells (Jackson et al., 2011), nor in 21 offspring that had been prenatally exposed. Some changes were noted in liver gene 22

expression profiles of female offspring. However, as in general the exposure of the fetuses 23 would be rather low, the observed alterations might have been caused as a secondary 24

response to the maternal inflammation in the lungs. 25

Shimuzu et al. (2009), from the same research group as Takeda et al. (2009) performed a 26

similar study in which pregnant mice were injected subcutaneously (100 µl of 1 mg/ml TiO2 27 solution) with nano-TiO2 (25-70 nm, anatase) on gestational days 6, 9, 12, and 15. This 28

study also investigated the effects of maternal exposure to nano-TiO2 on gene expression in 29

brain during the developmental period using cDNA analysis. Expression levels of the genes 30 associated with apoptosis were altered in the brain of newborn pups, whereas genes 31

associated with brain development were altered in early age. The genes associated with 32 response to oxidative stress were changed in the brains of 2 and 3 weeks old mice. Using 33

Medical Subject Headings (MeSH) terms information, the changes of the expression of 34 genes was found to be associated with neurotransmitters and psychiatric diseases. 35

In conclusion, although after inhalation or subcutaneous exposure of pregnant mice the 36 exposure of offspring in the uterus has been reported, exposure through this route is likely 37

to be low and some of the effects might be secondary to maternal toxicity induced by the 38

nanomaterials. The reported fetal effects were observed after high doses of intravenously 39 administered nano-TiO2, which are unlikely to occur in real life with the use of sunscreen 40

products. 41

42

SCCS Comment 43 No relevant study on reproductive toxicity is provided. One review article covering 44

exploratory studies has been provided (SI-II, Ema et al., 2010 (146)). Overall information 45 on this endpoint is as yet patchy and inconclusive. 46

47

1.5.9.1 Two generation reproduction toxicity 48

SCCS Comment 49

No data on two-generation reproductive toxicity is provided 50 51

52

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1.5.9.2 Teratogenicity 1

SCCS Comment 2

No data on teratogenicity is provided 3 4

5

1.5.10 Toxicokinetics 6

7 The following studies on toxicokinetics and metabolism have been provided: 8

Exploratory distribution, excretion study in rat 9 Reference: Fabian et al., Arch Toxicol. 2007 ref. No. 28 + 53; and 10

Fabian E. + Landsiedel R. ref. No. 28) 11

Guideline: Study considered a number of guidelines: EC Commission Directive 12 87/302/EEC (EC Commission Directive 1988), OECD Guidelines for 13

Testing of Chemicals (Method No. 417) (OECD Guidelines 1984), U.S. 14 EPA, Health EVects Guidelines, OPPTS 870.7485 (U.S. EPA 1998), and the 15

Japan/MAFF: Guidelines on the Compiling of Test Results on Toxicity 16 (Japan/MAFF2001). 17

18 Species/strain: male Wistar rat, 7–12 weeks old and weighed 200– 300g 19

20

Group size: 12 rats; 3 rats per group 21 Test substance: TiO2; 06/0489; P25 consisted of both anatase and rutile forms (70/30), 22

had no surface coating, the TiO2 primary particles were in the size range 23 20–30 nm; approximately 10 wt.% of the particle 24

agglomerates/aggregates are found in the nano-size range. BET specific 25 surface area of 48.6 m2/g. 26

Batch: 4165012298 (FI); 27

CAS No. 13463-67-7 28 Purity: unknown 29

30 Dose levels: 5 mg/kg body weight, TiO2 particles suspended in serum 31

Route: A single intravenous injection followed by biokinetics study 32 33

GLP: not applied 34 Study period: 35

36

Results 37 Analysis was performed on ICP-AES. According to the analytical method there were no 38

detectable levels of TiO2 in blood cells, plasma, brain, or lymph nodes. There were no 39 changes in the cytokines and enzymes measured in blood samples. Highest Ti retention was 40

observed in the liver at about 100-150 µg/g of organ with a limited clearance during the 41 next four weeks. Ti concentrations in spleen were only slightly lower than in the liver, but Ti 42

concentrations in kidneys and in lungs were about one order of magnitude lower with rather 43 remarkable clearance of about 66% during the next 14 days. 44

45

SCCS Comments 46

It is not clear which of the numerous noted guidelines were followed. Ti contents of the 47 organs were not corrected for background levels but untreated rats were analysed as well. 48

This means only 3 rats per group were analysed. Questions arise where the rest of the 49 administered TiO2 particles went, since an estimated dose of about 1.25 mg per rat were 50

injected and liver, spleen, lungs and kidneys amounted only to 600-700 µg per rat providing 51 no information on the remainder 500 µg. 52

53

Exploratory distribution, excretion study in rat 54

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1 Reference: Sugibayashi K., Todo H., Kimura E Safety evaluation of titanium dioxide 2

nanoparticles by their absorption and elimination profiles. Journal of 3 Toxicological Sciences 33(3), 293-8 (2008). 4

5 Guideline: not specified 6

Species/strain: mouse of unspecified strain 7 Group size: not clearly identified, probably 3-5 mice at each time point 8

Test substance: rod-shaped TiO2 rutile surface-coated with silica; (primary particle 9 diameter: 15 nm; agglomerated particle size: 220 nm); 10

Batch: HD-AW-150 from a Japanese company 11

Purity: rutile analysis by XRD, 27.5% silica content from surface modification, no 12 further analysis on impurities 13

14 Dose levels: no dose levels specified 15

Route: intravenous injection followed by biokinetics study in mice; 16 Administration: intravenous injection of titanium dioxide nanoparticles, single intravenous 17

injection; biokinetics after 5 min, 72 h and 30 d 18 GLP: not specified 19

Study period: 20

21 Results 22

Distribution of TiO2 (measured as Ti) was in blood and several tissues (primarily liver) but 23 not in brain. A slow decrease of TiO2 in liver was observed over time (~30% decrease in 24

one month). Observation of substantial amounts of Ti found in untreated mice prior to any 25 treatment due to significant natural food contamination; this led to an estimated dose of 90 26

µg/ Ti per day. After i.v. injection the Ti level was significantly increased in blood and 27 tissues. Ti concentrations per organs are provided but it is not clear whether or not these 28

were corrected for background Ti in all organs nor is the administered dose given. 29

30 SCCS Comment 31

Neither the strain nor the number of mice is clearly identified. The i.v. injected dose of TiO2 32 NP is also not specified. This study is therefore of no use to the current assessment. 33 34

35

Open literature 36

37

There are other toxicokinetic data of inhaled agglomerated TiO2 nanoparticles (Ma Hock et 38 al. 2008, 2009) showing oxidative stress and inflammatory reactions similar to previously 39

described 90-days exposure investigations. As far as toxicokinetic parameters were 40 evaluated, due to the detection limits, extrapulmonary TiO2 particles were not detected. 41

42 There are also toxicokinetic studies in which TiO2 NP were intravenously injected into the 43

vein of rodents (Fabian et al., 2007, and other papers). Retention was highest in the liver 44 followed by spleen, lungs, kidneys and it was highest at the first day compared to days 14 45

and 28. Cytokine levels remained unchanged indicating no detectable toxicity. 46

47 There are no new toxicokinetic data on the absorption of TiO2 NP after administration to the 48

gastrointestinal-tract (GIT). The most recent study from Wang et al. 2008 used 49 unrealistically high doses of 5 g/kg BW in rats such that their findings are not useful and 50

may even be modulated by uncontrolled other forms of intake like inhalation of aspiration. 51 Their biodistribution data showed the highest retention in the liver followed by spleen, 52

kidneys and lungs. Thus toxicokinetics data after GIT administration still rely on the studies 53 of the group of Alexander Florence, in the 1990ies. These suggest that about 5-7% of the 54

administered 500nm TiO2 particles were absorbed and retained in the body, mainly in the 55

liver. 56 57

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Applicant’s conclusions 1 Intravenous administration of large doses of nano TiO2 did not result in adverse effects or 2

signs of toxicity in rodents. A non-specific and expected tissue distribution of TiO2 was 3 observed. No TiO2 was detected in brain, and the levels in other organs decreased over 4

time. 5 6

SCCS Comment 7 The limited available evidence suggests that if TiO2 nanoparticles become systemically-8

available, they may accumulate mainly in liver with a very slow clearance. 9 10

11

1.5.11 Photo-induced toxicity 12

13

1.5.11.1 Phototoxicity / photoirritation and photosensitisation 14

15

Photo- irritation 16

Guidelines: OECD good laboratory principles 17

Product tested: TiO2 T805 (1992 batch 030492) 18

Species: SPF NZ white rabbits (Ch. River), Female 19

Groups: 3 animals/group 20

Dosing: 3, 10, 30% in ethanol 96% during 100 min 21

Exposure area: 15 – 7.5 cm total, each exposure side spot approximately 2 cm 22

diameter 23

UVA-light: 310-420 nm peak 365nm total dose 10J/cm² (approx. 50 min dosing) 24

Readings: 30 min, 24h, 48h, 72 h after UV-exposure 25

Observations: No irritation found, neither non-irradiated as irradiated TiO2 treated 26

animals. 27

Reference: 15 28

Conclusion: TiO2 (T805) is not photo-irritating for rabbit skin under the assay 29

conditions after UVA irradiation up to 10 J/cm². 30

31

32

Guidelines: OECD good laboratory principles 33

Product tested: TiO2 T805 (1992 batch 030492) 34

Species: SPF albino guinea pigs (Ch. River) 35

Sex: Males & Female 36

Experimental protocol: Following Ichikawa, Armstrong & Harber 1981, Induction treatment 37

followed by challenge 12 days later 38

Groups: Test groups 5 animals of each sex 39

Dosing: 30% TiO2 in ethanol (96%) at induction treatment & challenge 40

treatment (day 12) 41

Induction protocol: 42

- 6-8 cm area cleared from fur 43

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- Area is subcutaneously pre-treated with Freund adjuvant and exposed to 0.2 ml of 1 suspension followed by UVA-light: 310-420 nm (peak 365 nm) total dose 10 J/cm² 2

- In total 5 treatment over 2 weeks (only first time Freund adjuvant was used). 3

- Skin was not cleared after treatment 4

- Reading after each treatment 5

Challenge protocol: 6

- 12 days after last induction 7

- 5-10 cm area cleared from fur 8

- Exposed to 0.5 ml of 30% TiO2 (T805) suspension direct followed by light: 310-420 nm 9

(peak 365 nm) total dose 10 J/cm² - 37 min 10

Observations: No irritation found, neither during induction phase or challenge 11 phase, in both non-irradiated as irradiated TiO2 treated animals. 12

Conclusions: TiO2 (T805) is not photo-sensitizer for guinea pigs under the assay 13

conditions after UVA irradiation up to 10 J/cm². 14

Reference: 17 15

16

Human data: 17

Product tested: 0685115 (No other info in the document) 18

Species: 60 volunteers (19-77 y) of which 50 completed the study 19


Protocol: 21

- Induction: 3 patches per week (Mon, Wed, Fri) during 3 weeks (0.2 ml TiO2 suspension 22

per patch – no concentration reported). Patches remain at place 24 h (removal by 23 volunteers). If reaction, next patch was moved to adjacent area (testing was 24

discontinued if severe reaction was noted) 25

- Challenge: 2 weeks after last induction at different spot 26

Result: No effects observed, in any of the volunteers 27

Conclusion: Product 0685115 is not a sensitizer for humans under the assay 28

conditions 29

Reference: 27 30

31

SCCS Comment 32

The study is not a photosensitisation study but is only sensitization study. 33 34

35

Product tested: 0685115 (No other information in the document) 36

Species: 29 human volunteers (18-60 y) of which 25 finished the whole study 37

(drop-out were not related to the test) 38


Pre-testing: MED (Minimal Erythemal Dose) of unprotected skin of each volunteer 40 was assessed. [MED = time interval or dose of UV sufficient to 41

produce minimal perceptive erythema] 42

Light source: UV A (320-400 nm), 3 min(approximately 10.08 Joules) 43

Protocol: 44

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- Induction: 2 spot prepared for exposure to compound 0685115, of which one is 1 irradiated while the other is not be irradiated. 2

The areas cleared from hair of 1 inch/ 1 inch, and 0.2 ml (no concentration of TiO2 3

suspension reported) of test material is placed on the spots. Exposed side is kept under 4 patch during 24 h. 5

2 applications applied per week for 3 weeks (total 6 applications). 6 After removal of patch spots irradiated with a dose of 2x MED of the volunteer 7

- Challenge: 2 weeks after last induction at different spots on the back. 8

Spots are under patch for 24 h, then irradiated for 3 min (non erythemogenic dose). 9 Reading after 24, 48 & 72 h 10

Result: No effects observed, in any of the volunteers 11

Conclusion: Product 0685115 is not a photo-sensitizer for humans under the assay 12

conditions 13

Reference: 28 14

15

SCCS Comments 16 Ref 16 and 18 could not be found. The given references are not correct, as they do not 17

report photo-irritation (Sonnenschutzformulierungen: Lotions und Cremes) 18 19

1.5.11.2 Phototoxicity / photomutagenicity / photoclastogenicity 20

21

A number of studies has not been reviewed as part of this assessment, because the 22

experiments were performed with bacterial cells. As discussed in section 3.3.6, bacterial 23 mutagenicity assays are not considered to be appropriate for the testing of nanoparticles 24

compared to mammalian cell systems. Other studies were not reviewed because they are 25 related to test materials that are either not nanomaterials, or they lack data on material 26

characterisation to establish whether they were relevant nanomaterials to this assessment. 27 28

Phototoxicity test in vitro 29

Guideline/method: OECD TG432 30

Test system: Balb/c 3T3 fibroblasts, neutral red uptake (NRU) 31

Replicates: no replicates 32 Test item: T805 (coated, A/R, PSMA 1 type), T817 (coated, A/R, PSMA 1 type), 33

TiO2 P25 (non coated) 34 Batch: 05 10067 (T805), 04095 (T817), P1S-3087 (p25) 35

Vehicle: EBSS wit 1% ethanol 36 Concentrations: 0.78 t0 1—mg/L UV-A: 5.0 J/cm² 37

Exposure: 0, 0.79, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L 38 Negative control: vehicle 39

Positive control: not included 40

GLP: no 41 Date of report: 1999 42

Reference: Submission DHS, 24 and 25 43 Balb/c 3T3 cells were pre-incubated with eight different concentrations (0.79, 1.56, 3.13, 44

6.25, 12.5, 25, 50 and 100) of the nanoparticles in two 96-well plates, one plate was 45 subsequently exposed to 5 J/cm2 UVA while the other plate was kept in the dark. Medium 46

was then replaced and after 24 h cell viability was determined by spectrophotometrical 47 evaluation of neutral red dye uptake (3 h incubation of neutral red). The phototoxic 48

potential was determined by calculation of the ratio of the nanoparticle concentration that 49

reduced viability by 50% (NR50) in presence versus absence of UV irradiation. 50

Results 51

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T805 and T817 showed neither cytotoxicity nor phototoxicity up to a concentration of 1 100mg/L. The p25 (non coated NP) sample also was not cytotoxic up to the highest 2

concentrations, but in the presence of irradiation a viability reduction of 82 % (at 50 mg/L) 3 and 44% (at 100 mg/ml) was observed. 4

5

Conclusion 6

p25 sample is phototoxic towards Balb/c 3T3 cells, while T805 is not phototoxic. 7 8

SCCS Comment 9 This study is indicative of the importance of coating on the phototoxic properties of TiO2 10

nanoparticles. 11

12 13

Photoclastogenicity test in vitro 14

Guideline/method: Chromosomal aberration test in presence or absence of UV treatment 15

Test system: CHO-WBL cells 16 Replicates: Duplicate 17

Test item: See table 18 Batch: - 19

Vehicle: Ethanol (sample A), PBS (samples B and C), DMSO (D, E,F,G and H) 20

Concentrations: Three concentrations for each sample with as higest concentration either 21 5000 µg/ml or a dose that resulted in less than 50% cytotoxicity 22

Exposure: 3 h followed by 17 h recovery 23 UV dose: 750 mJ/cm2 (provided 15 min after NP treatment initiation) 24

Negative control: Vehicle 25 Positive control: 8-methoxypsoralen (8-MOP), 4-nitroquinoline-1-oxide (NQO) 26

GLP: - 27 Published: yes 28

Reference: Theogaraj et al., 2007 29

30

Test items used: 31

32

The photoclastogenicity of TiO2 was determined in CHO cells. S9 mix was not included in 33 the protocol. Cells were treated in the dark for 15 min and then UV radiated. After 34

irradiation the cultures were incubated in the dark, after which the medium was removed. 35

Cultures were washed and fresh medium was added for a further 17 h. Cells were then 36 harvested and stained slides were then evaluated for the presence of chromosomal 37

aberrations. 38

39

Results 40

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No increases in chromosomal aberration frequencies were found either in the presence or 1 absence of UV up to the highest treatment concentrations. 2

3

Conclusion 4

No photogenotoxicity was observed under the applied testing conditions. 5 6

SCCS Comment 7 Uptake of the NP into the cells was not evaluated. The UV treatment was performed shortly 8

after initial exposure to the particles (15min). At this time uptake may have been limited. 9 10

11

1.5.12 Human data 12

A number of human studies have been quoted on different versions of skin patch test. Some 13

of the studies have used TiO2 materials for which no information on material 14 characterisation has been provided, whist others have been reviewed in relevant sections. 15

16

1.5.13 Special investigations 17

A number of studies have been provided, relating to cytotoxicity, coating stability and 18 photostability of TiO2 materials. Many of these studies have used TiO2 materials for which 19

information on material characterisation has not been provided. 20

21

1.5.14 Human safety evaluation (including calculation of MoS) 22

23 Given the very low, if any, dermal penetration of nano-TiO2 when applied on skin, and in 24

consideration of the low toxicity observed, the calculation of a margin of safety (MoS) is not 25 relevant for this assessment. 26

27 Any exposure to nano-TiO2 via oral route from a dermally applied product is also likely to 28

be insignificantly low. Again in consideration of the low toxicity observed, the calculation of 29

a margin of safety (MoS) for the oral route is not relevant. 30 31

In view of the concerns over safety of nano-TiO2 via inhalation route, its use in applications 32 that might lead to inhalation exposure (such as powders or sprayable products) is not 33

recommended and therefore has not been considered in the calculation of MoS. 34 35

1.5.15 Discussion 36

37

General considerations: 38

The submission consists of fifteen (15) TiO2 nanomaterials that vary in terms of various 39 physicochemical parameters. The studies provided in support of the submission range from 40

old to recent ones. A major proportion of the (old) studies are on materials for which little 41 or no information on characterisation has been provided, which makes it difficult to relate 42

many of them to the nanomaterials under current assessment. 43 44

The evaluation by the SCCS of these and other studies provided in this submission has 45 shown that many of them are not relevant to the nanomaterials in the submission. 46

Therefore the relevance and usefulness of the data provided for this evaluation is poor and 47

patchy. It is difficult (in some cases impossible) to relate the studies to the types of 48 nanomaterials under evaluation. It would have been more productive if a complete set of 49

supporting data was provided on one (or a few) rather than several different TiO2 50 nanomaterials in a single submission. 51

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1 Physicochemical properties: 2

- The studies provided in the submission relate to a range of TiO2 materials that comprise 3

micronized, ultrafine, or nano-sized particles. The physicochemical characterisation data 4 include coated and non-coated materials, composed of rutile and/or anatase forms of 5

TiO2. On the basis of the physicochemical data provided, the SCCS has considered the 6 materials in three broad groups on the basis of crystalline form and photocatalytic 7

activity. 8

- The SCCS agrees that TiO2 nanoparticles, due to agglomerative behaviour, are likely to 9

be present in the final sunscreen products mainly in the form of agglomerates, which 10

can also be in the nanoscale. It can therefore be assumed that the consumer is likely to 11 be exposed mainly to TiO2 agglomerates. However, it is also possible for the 12

agglomerates to de-agglomerate under certain conditions of formulation/use. Therefore, 13

the SCCS has considered the size of the primary particles more important than the size 14 of agglomerates for the purposes of risk assessment. 15

- As nanoparticles may have different properties and biokinetic behaviour than their 16

soluble equivalents, it is important to know the exact purity/impurity profile of a 17 nanomaterial intended for use in a cosmetic product (SCCS Guidance, SCCS/1484/12). 18

This opinion therefore does not cover TiO2 nanomaterials that have TiO2 purity less than 19 99%, and for which an acceptable impurity profile has not been provided. The opinion 20

may, however, be also applicable to other TiO2 nanomaterials that are similar to the 21 nanomaterials in this opinion in terms of the physicochemical parameters listed in Tables 22

1-3, and other specific provisions laid out in Section 2. 23

- None of the materials evaluated in the submission is comprised of completely spherical 24

particles because their reported aspect ratios are >1.0. However, the SCCS has 25

accepted an aspect ratio range between 1.0 and 4.5 on the basis that a lower aspect 26 ratio particle is less likely to be of a concern compared to higher aspect ratio ones. 27

- Zeta potential measurements have been provided for some materials, and not for others 28 due to difficulties in measuring zeta potential for hydrophobic nanomaterials. 29

- Among the nanomaterials assessed, the SCCS has noted a potential concern in relation 30 to photocatalytic activity, and stability of the coating, of some of the materials. It is 31

stated by the Applicant that all coatings on the materials included in the submission are 32 stable. Three (3) studies have been provided, which show that coatings are stable. 33

However, from the other physicochemical data provided, it is less clear how stable the 34

coatings are in final formulations. The photocatalytic activity data, which is measured in 35 formulations, clearly indicate that either some of the materials were not completely 36

coated, or some of the coatings (e.g. organic, organosilanes) were not so stable in the 37 formulations. This is an important aspect to ascertain because application of a 38

formulation containing a nanomaterial that has a significant photocatalytic activity may 39 lead to local effects on sun-exposed skin. Such effects may or may not manifest during 40

the immediate use, and it is important to investigate the possibility of latent effects 41 following the use of a skin product that contained photocatalytic nanoparticles. This is 42

because, whilst most studies on dermal absorption indicate that TiO2 nanoparticles are 43

not able to penetrate the skin deep enough to reach live cells of the epidermis/dermis, 44 they do show that nanoparticles can penetrate into stratum corneum, and can also enter 45

hair follicles and sweat glands. It is therefore possible that a trace amount of 46 nanoparticles may remain embedded in stratum corneum, in hair follicles, and/or sweat 47

glands, potentially over several days after skin application of a product and washing off. 48 If the nanoparticles have a significant photocatalytic activity, there is a possibility that 49

they may cause generation of reactive radical species on exposure to sunlight, long after 50 the skin formulation had been applied and washed off. This, in a close proximity of living 51

cells, raises a concern over the possibility of harmful effects. Generally metal(oxide) 52

nanomaterials which exhibit a high photocatalytic activity are those that are either 53 uncoated, partially coated, or have not been quenched by other means (e.g. doping) to 54

adequately reduce photoreactivity. The TiO2 nanomaterials in the current submission 55

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that have a high photocatalytic activity include anatase materials in uncoated (S75-G) 1 and coated forms (S75-F, S75-O). Three (3) other rutile coated nanomaterials also have 2

comparatively lower but still significant levels of photocatalytic activity (S75-C, S75-D, 3 S75-E). 4

- The SCCS considers up to 10% photocatalytic activity compared to corresponding non-5 coated or non-doped reference as acceptable. 6

- In view of this, the SCCS does not recommend the use of nanomaterials that have a 7

high photocatalytic activity (S75-F, S75-G, S75-O) in dermal formulations. These 8 materials can only be recommended after appropriate coating/doping has been applied 9

to quench their photocatalytic activity down to acceptable levels. 10

- Three rutile materials (S75-C, S75-D, S75-E) with relatively lower but still significant 11

levels of photocatalytic activity may be used in dermal formulations, but further 12

investigations over longer post-application periods may be necessary to ascertain that 13

they do not pose a risk due to photocatalytic activity. 14

Acute toxicity: 15

- The studies provided on acute oral toxicity in the submission mainly relate to TiO2 16 nanomaterials that are anatase/rutile mixtures, coated with trimethoxy-n-octyl-silane. 17

From the limited relevant information provided, and considering that oral intake is not 18 likely to be the major route of exposure to TiO2 nanomaterials from dermal application 19

of formulations, the acute oral toxicity of TiO2 is unlikely to be of a concern. 20

- The studies provided on acute dermal toxicity relate to an ultrafine TiO2 material and a 21

material described as ‘natural colour’, and are therefore of no relevance to the 22

assessment of nanomaterials. 23

- No study has been provided on acute inhalation toxicity. Sub-chronic (inhalation) and 24

chronic (instillation) studies have indicated substantial inflammatory responses and 25 overload associated with diminishing particle clearance in a dose dependent manner, 26

and histological indications of epithelial hypertrophy and hyperplasia. 27

- The limited relevant information provided in the submission, and other information in 28

the open literature, indicates that TiO2 nanomaterials are likely to be non-toxic via oral 29 or dermal application routes. However, inhalation exposure to TiO2 nanoparticles is 30

likely to cause substantial inflammatory effects in the lung. 31

32 Skin irritation: 33

- Only two of the studies provided are relevant to the TiO2 nanomaterials. They relate to 34 anatase/rutile mixtures, coated with trimethoxy-n-octyl-silane. The results showed 35

primary irritation index between zero and 0.3. Two studies using ultrafine grade 36 materials showed the mean irritation scores of 0.3 and 1.58-1.92 during 5 day repeat 37

applications on rabbit skin. Other studies also showed the tested materials to be either 38 mild- or non- irritant to rabbit and guinea pig skin, but it is not clear whether the tested 39

materials were nanomaterials. 40

- From the limited relevant information, it can be considered that TiO2 nanomaterials are 41 likely to mild- or non- irritant to skin. 42

43 Eye irritation: 44

- Two studies tested TiO2 anatase/rutile mixtures, coated with trimethoxy-n-octyl-silane. 45 From the studies, the derived primary irritation index was between zero and 0.3. A 46

different study used ultrafine rutile material coated with alumina/silica and regarded the 47 tested material as slightly irritant to rabbit eye. Another study found the tested TiO2 48

materials to be moderately irritant to rabbbit eye, but it is not clear whether the 49

material was a nanomaterial. 50

- From the limited relevant data provided, eye irritation potential of nano-TiO2 appears to 51

be low. 52

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1 Skin sensitisation: 2

- Two of the provided studies have regarded TiO2 nanomaterials (anatase/ rutile mixture, 3 coated with trimethoxy-caprylylsilane or trimethoxy-n-octyl-silane) as non-sensitiser. 4

Another ultrafine material (rutile, coated with alumina/silica) is classified as a weak 5 sensitiser, but characterisation data (particle size distribution) has not been reported to 6

indicate what proportion of the particles was in the nano-scale. 7

- Due to the absence of skin penetration of TiO2 as demonstrated by many studies 8

included in this dossier, the usefulness of the Buehler test for assessing sensitisation 9 potency of nanomaterials is doubtful as it is based on exposure to intact skin. 10

- From the limited relevant data provided, TiO2 nanomaterials appear to be non- or weak 11

skin sensitisers. 12

13

Dermal absorption: 14

- A number of in vitro and in vivo dermal penetration studies have been provided with the 15

submission. In addition, there is a body of open literature on this subject. The evidence 16

from these studies supports the conclusion that TiO2 nanoparticles are unlikely to 17 penetrate across the skin to reach viable cells of the epidermis. In these studies, TiO2 18

nanoparticles have been shown to penetrate only to the outer layers of the stratum 19 corneum, and there is as yet no conclusive evidence to show that they do reach living 20

cells of the epidermis/dermis. Studies have also shown that TiO2 nanoparticles do not 21

penetrate the (simulated) sunburnt skin. 22

- Despite the extensive database showing a general lack of TiO2 nanoparticle absorption 23

via the dermal route, there are a few gaps in the knowledge. For example, it is not clear 24

whether TiO2 nanoparticles will be able to penetrate through cuts and bruises, or over 25 repeated or long term applications of a sunscreen formulation. 26

- A number of studies have indicated that TiO2 nanoparticle can enter the hair follicles 27

and sweat glands, and that they may remain there for a number of days. This is a 28 scenario in which TiO2 nanoparticles are likely to get and remain in a close proximity to 29

the living cells for a length of time. A photocatalytic nanoparticle in such a situation may 30 cause generation of reactive oxyradical species (ROS) and potential harmful effects 31

when exposed to sunlight. As mentioned before, more data would be needed to justify 32 the use of those TiO2 nanoparticles in skin applications that have a considerable level of 33

photocatalytic activity. 34

35 Repeated dose toxicity: 36

- Only two of the four provided subchronic studies on repeated dose toxicity are relevant 37 to the TiO2 nanomaterials under evaluation. However, these studies relate to oral 38

exposure only, from which a LOAEL of 5 mg/kg bw/d has been derived. 39

- No chronic toxicity study (>12 months) is provided, although a chronic inhalation study 40

has been provided (Section 3.3.1.3). 41

42 Inhalation toxicity: 43

- Studies in open literature indicate that subacute repeated dose respiratory toxicity 44

studies with nano size TiO2 induce an acute inflammation in the lungs that may be 45

reversible depending on the dose and the time evaluated after exposure. In view of this, 46 acute inflammation (spray) applications, which may result in inhalation exposure is not 47

recommended by the SCCS. 48

49

Mutagenicity/ Genotoxicity: 50

- Although an extensive range of studies on mutagenicity has been provided in the 51

submission, most of them have not been conducted in any special consideration of the 52

nano-related properties of the test materials. 53

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- Several studies have been performed mainly to investigate mechanistic effects relating 1 to DNA damage and genotoxic properties. These studies are usually not performed 2

according to specific genotoxicity guidelines (e.g. OECD). Many of the studies have not 3

evaluated the effects in a dose- and/or time- dependent manner. Those that have 4 addressed this, often reveal no clear dose- or time- dependent effects. 5

- From the provided studies, and open literature, TiO2 particles have also been 6

reported, or suggested, to interfere with the assays, because: 7

- Micronucleus scoring is difficult in the presence of TiO2 particles. This effect 8

was suggested to explain for the occasionally observed decreases in MN counts 9 after TiO2 treatment (Falck et al., 2009). 10

- It has been suggested (although not shown) that artefacts may be caused in 11 relation to the use of cytochalasin B for micronucleus testing. On one hand, it is 12

suggested that nanoparticles may interfere with cytochalasin B (binding), and 13

on the other, that the cytochalasin B may act as an inhibitor of the uptake of 14 nanoparticles in cells potentially leading to false negatives (Landsiedel et al., 15

2010). 16

- Due to the current lack of information on the possible cellular uptake and 17

subsequent translocation of TiO2 nanoparticles to nucleus, it is not possible to 18 draw a conclusion on whether or not exposure to TiO2 nanomaterials can lead 19

to mutagenic effects. 20

- Overall in a number of assays, TiO2 nano particles were observed to induce DNA 21

damage, so TiO2 nano particles have to be considered genotoxic. 22

- It is also of note that appropriate coating of nanomaterial to quench surface 23 photocatalytic activity will also reduce the likelihood of generation of reactive oxygen 24

species (ROS), which may in turn reduce the chances of genotoxicity. 25

26

Carcinogenicity: 27 - Pigmentary and ultrafine TIO2 materials have been tested for carcinogenicity by oral 28

administration in mice and rats, by inhalation exposure in rats and female mice, by 29 intratracheal administration in hamsters and female rats and mice, and by subcutaneous 30

injection in rats and by intraperitoneal administration in male mice and female rats. 31

- According to the evaluation of TiO2 by IARC (2010), induction of lung tumours was 32 observed in two inhalation studies with rats. Two other inhalation studies in rats, and 33

one in female mice gave negative results. Intratracheally instilled female rats showed 34 an increased incidence of lung tumours following treatment with two types of titanium 35

dioxide. Tumour incidence was not increased in intratracheally instilled hamsters and 36 female mice. Oral, subcutaneous and intraperitoneal administration did not produce a 37

significant increase in the frequency of any type of tumour in mice or rats. IARC 38 concluded that there is inadequate evidence in humans for the carcinogenicity of 39

titanium dioxide but sufficient evidence in experimental animals for the carcinogenicity 40

of titanium dioxide. Both nano and non nano size Titanium dioxide was classified as a 41 Group 2B carcinogen (Possibly carcinogenic to humans). 42

- In their recent evaluation of TiO2 NIOSH has determined that ultrafine TiO2 which 43 contains nano-sized TiO2 is a potential occupational carcinogen and, that there is 44

insufficient data to classify fine TiO2 as potential occupational carcinogen after inhalation 45 (NIOSH 2011). 46

- Nano titanium dioxide has been studied in 2 two-stage skin carcinogenicity studies with 47 mice, 2 two-stage skin carcinogenicity studies with rats, and one two-stage lung study 48

with rats. Both noncoated (ncTiO2) and coated titanium dioxide have been studied in the 49

two-stage mouse skin carcinogenicity studies with CD1 mice and a transgenic mouse 50 strain (rasH2). In one well performed study with non-coated and alumina and stearic 51

acid coated TiO2, no promoter activity was found (Furukawa et al., 2011). Promoter 52

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activity was also not found for ncTiO2 in the other study (Sagawa et al., 2012). 1 However, it is difficult to draw a firm conclusion from this study with silica coated 2

titanium dioxide due to lack of positive controls and very high tumour incidence in the 3 ‘initiated’ mice. 4

- Non-coated titanium dioxide was studied in 2 two-stage rat skin carcinogenicity studies. 5 Although, no tumour promoter activity was observed, it is difficult to draw any 6

conclusion since little experience with the model used is available and no positive 7 controls have been used in the studies. 8

- One, two-stage rat lung carcinogenicity study has been carried out with non coated 9 titanium dioxide. The rats were ‘initiated’ by DHPN in the drinking water prior to intra-10

pulmonary spraying with ncTiO2. The experiment demonstrated promoter activity of 11

ncTiO2 (Xu et al., 2011). 12

- Since TiO2 particles have shown carcinogenic activity (after inhalation) and since nano 13

ncTiO2 showed promoter activity after intra-pulmonary spraying, the use of nano TiO2 14 in sprayable applications is not recommended by the SCCS. 15

16 Reproductive toxicity 17

- No study has been provided on reproductive toxicity that is relevant to the 18

nanomaterials under assessment. A review article covering exploratory studies in mice 19 has been provided, which relates to the use of a TiO2 material which is <10µm (with no 20

further information), and a TiO2 nanomaterial with primary particle size 25-70 nm (no 21

further information). 22

- Other studies in open literature have indicated the possibility of placental transport in 23

pregnant animals into the foetus, or found effects in the offspring for various 24

manufactured nanomaterials including nano-TiO2. However, the information relating to 25 this endpoint is patchy and therefore inconclusive. 26

27 Photo-induced toxicity 28

- Only a few studies have been provided that are relevant to the nanomaterials under 29 assessment. 30

- These indicate that TiO2 materials may not be photo-sensitisers. However, concerns 31

regarding the use of photocatalytic nanomaterials in dermal formulations discussed 32 above need to be taken into consideration. 33

- Several studies have specifically addressed photo-sensitization effects TiO2. However, 34 the outcomes of these studies need to differentiate between photo-sensitization and 35

other local effects on skin (taking into account the aspect of penetration), versus 36 potential effects at other target sites. 37

38

Toxicokinetics: 39

- Two studies have been provided in the submission on toxicokinetics of TiO2 following 40

intravenous injection in rats and mice. In addition, there are few other relevant studies 41

in the open literature relating to inhalation and intravenous, as well as limited 42 (questionable) information on oral administration routes. 43

- The available evidence suggests that, if TiO2 particles become systemically available by 44 the oral and inhalation uptake pathway, they are likely to accumulate mainly in the liver, 45

followed by a very slow rate of clearance. 46

47

Special investigations: 48 No relevant specific studies have been provided apart from those already discussed above 49

under relevant endpoints. 50 51

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1

2

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2. CONCLUSIONS 1

This opinion is based on the risk assessment of nano-sized titanium dioxide (TiO2) for use 2

as a UV filter in sunscreen formulations. It is important to note that risk assessment of 3 nanomaterials in general still has certain gaps in the knowledge - for instance in relation to 4

the behaviour of nanoparticles in a test medium, or in the animals. This has led to 5 uncertainties over whether the nanoparticles are able to reach and interact with various 6

moieties and biological target sites, and whether, on dermal application, they may penetrate 7 through damaged skin, or during repeated or long term applications. There are also 8

uncertainties over the validity of the currently available tests used for nanomaterials. 9 However, a positive toxic response in these tests is still considered valid for risk assessment 10

as it would indicate a hazard potential. 11

As discussed above, the safety data provided in support of the fifteen (15) nanomaterials is 12 quite patchy, and is only partially useful for any of the given nanomaterials. However, the 13

SCCS took the view that this submission could be considered for evaluation as an exception. 14 This is because some additional information on TiO2 nanomaterials is available in open 15

literature which is relevant for this evaluation. Also, for example, although the safety data 16 provided in the submission on rutile nanomaterials is insufficient, the studies on anatase 17

form (or rutile/anatase mixtures) could be considered as a surrogate because published 18 studies in open literature have regarded anatase a greater safety concern than the rutile 19

form. However, as the evaluation is still based on limited information which could be related 20

to specific nanomaterial types in the submission, this opinion is limited to the nanomaterials 21 indicated below: 22

23 - On the basis of physicochemical considerations discussed above, this opinion applies to 24

the TiO2 nanomaterials presented in this submission. In addition, the opinion may also 25 be applicable to other TiO2 nanomaterials that are similar to the nanomaterials covered 26

in this opinion in terms of physicochemical parameters listed in Tables 1-3, and the 27 specific provisions laid out in the overall conclusions below. 28

- It needs to be stressed that the main consideration in the current assessment is the 29

apparent lack of penetration of TiO2 nanoparticles through skin, which is supported by a 30 body of evidence both in the form of studies provided by the Applicant and other studies 31

reported in open literature. In the absence of a systemic exposure, a margin of safety 32 (MoS) could not be calculated for TiO2 nanomaterials in this assessment. From the 33

limited relevant information provided in the submission, and the information from open 34 literature, the SCCS considers that TiO2 nanomaterials in a sunscreen formulation are 35

unlikely to lead to: 36

o systemic exposure to nanoparticles through human skin to reach viable cells of 37

the epidermis, dermis, or other organs; 38

o acute toxicity via dermal application or incidental oral ingestion. This, however, 39 does not apply to sprayable applications that may lead to inhalation exposure of 40

TiO2 nanomaterials, which may result in lung inflammation; 41

o skin irritation, eye irritation, or skin sensitisation when (repeatedly) applied on 42

healthy skin (except possible photoxicity of insufficiently coated nanomaterials); 43

o reproductive effects when applied on healthy skin. 44

- Some TiO2 nanoparticles have been shown to be able to damage DNA and should be 45 considered genotoxic. However as negative results have also been reported, the current 46

evidence in relation to potential genotoxicity of TiO2 nanomaterials is not conclusive. 47

TiO2 particles have also shown to lead to carcinogenic effects after inhalation. These 48 manifestations are a major hazard concern. However, no penetration was found through 49

the stratum corneum of reconstructed human full thickness skin models and no DNA 50 damage was detected by the Comet assay in these cells in contrast to epidermal cell 51

line. Considering the absence of a systemic exposure, the SCCS considers that the use 52

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of nano TiO2 in dermally applied cosmetic products should not pose any significant risk 1 to the consumer. 2

- Evidence on acute and sub-chronic inhalation toxicity does not support the overall safety 3 of use of TiO2 nanomaterial formulations for spray applications. In addition, tumour 4

promoter activity of nano (non-coated) TiO2 has been shown after intra-pulmonary 5 spraying. Therefore the SCCS does not recommend the use of nano TiO2 in sprayable 6

applications. This may be reconsidered if further evidence is provided to rule out the 7 possibility that the nanoparticles can reach the lower respiratory tract during spray 8

applications. 9

- Although there is no conclusive evidence at present to indicate penetration of TiO2 10

nanoparticles through the skin to viable cells of the epidermis, a number of studies have 11

shown that they can penetrate into the outer layers of the stratum corneum, and can 12

also enter hair follicles and sweat glands. It is therefore recommended not to use TiO2 13 with substantially high photocatalytic activity (e.g. S75-F, S75-G, S75-O) in sunscreen 14

formulations. Other TiO2 nanomaterials that have a relatively lower but still significant 15 level of photocatalytic activity (e.g. S75-C, S75-D, S75-E) may be used, but further 16

investigations over longer post-application periods taking into account the potential 17 photocatalytic activity post-application, whilst allowing for appropriate lag-time and 18

using realistic application scenarios may be necessary to ascertain that they do not pose 19 a risk due to photocatalytic activity. 20

21

Overall conclusion 22

1. Does SCCS consider that use of titanium dioxide in its nanoform as an UV-filter in 23

cosmetic products in a concentration up to maximum 25.0 % is safe for the consumers 24 taken into account the scientific data provided? 25

26 On the basis of the available evidence, the SCCS has concluded that the use of TiO2 27

nanomaterials with the characteristics as indicated below, at a concentration up to 25% as a 28 UV-filter in sunscreens, can be considered to not pose any risk of adverse effects in humans 29

after application on healthy, intact or sunburnt skin. This, however, does not apply to 30

applications that might lead to inhalation exposure to TiO2 nanoparticles (such as powders 31 or sprayable products). Furthermore, this assessment applies to the TiO2 nanoparticles 32

presented in the submission, but may also be applicable to other TiO2 nanomaterials that 33 are similar to the parameters in Tables 1-3, i.e. TiO2 nanomaterials that: 34

have TiO2 purity of ≥99%, or in case of a lesser purity, the impurities must be 35 demonstrated to be safe for use in cosmetic formulations; 36

are composed of mainly the rutile form, or rutile with up to 5% anatase, with 37 crystalline structure and physical appearance as described in the current submission, 38

i.e. clusters of spherical, needle, or lanceolate shapes; 39

have a median particle size based on number size distribution of 30 to 100 nm 40 (measured by different methods) as submitted in the dossier, or larger. Thus whilst 41

primary particle size may be smaller (around 10 nm), the median particle size of TiO2 42 nanomaterials in a cosmetic formulation must not be smaller than 30 nm in terms of 43

number based size distribution; 44

have an aspect ratio from 1.0 and up to 4.5, and volume specific surface area up to 45

460 m2/cm3; 46

are coated with one of the coating materials described in Table 1, and the coatings are 47

stable in the final formulation and during use. Other cosmetic ingredients applied as 48

stable coatings on TiO2 nanomaterials can also be used, provided that they can be 49 demonstrated to the SCCS to be safe and the coatings do not affect the particle 50

properties related to behaviour and/or effects, compared to the nanomaterials covered 51 in this opinion. 52

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are photostable in the final formulation; 1

do not have photocatalytic activity. However, the SCCS considers up to 10% 2

photocatalytic activity compared to corresponding non-coated or non-doped reference 3 as acceptable. 4

It is also worth highlighting again that this opinion is based on the currently available 5 scientific evidence which shows an overall lack of dermal absorption of TiO2 nanoparticles. 6

If any new evidence emerges in the future to show that the TiO2 nanoparticles used in a 7 sunscreen formulation can penetrate skin (healthy, compromised, or damaged skin) to 8

reach viable cells, then the SCCS may consider revising this assessment. 9

It should also be noted that the risk assessment of nanomaterials is currently evolving. In 10

particular, the toxicokinetics aspects have not yet been fully explored in the context of 11

nanoparticles (e.g. the size dependency). Also, long term stability of the coatings remains 12 unclear. At the moment, testing of nanomaterials and the present assessment, are both 13

based on the methodologies developed for substances in non-nano form, and the currently 14 available knowledge on properties, behaviour and effects of nanomaterials. This assessment 15

is, therefore, not intended to provide a blue-print for future assessments of other 16 nanomaterials, where depending on the developments in methodological risk assessment 17

approaches and nano-specific testing requirements, additional/different data may be 18 required and/or requested on a case-by-case basis. 19

It is also important to note that the potential ecotoxicological impacts of nano TiO2 when 20

released into the environment have not been considered in this opinion. 21

22

2. In order for the COM to differentiate in the regulation between materials in its nanoform 23 and its non-nano form, can the SCCS give quantitative and qualitative guidance on how this 24

differentiation should be given based on the particle size distribution or other parameters? 25

A detailed SCCS guidance on risk assessment of nanomaterials in cosmetics has recently 26

been published (SCCS/1484/12). The guidance provides a detailed account of the important 27 nano-related parameters that should be considered in relation to physicochemical 28

characterisation, hazard identification, exposure assessment and risk assessment of 29

nanomaterials. 30

31

3. MINORITY OPINION 32

/ 33

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ABBREVIATIONS AND GLOSSARY OF TERMS 1

2 BET Brunauer-Emmett-Teller method based on nitrogen gas absorption 3

CAS A chemical registry system established by the Chemical Abstracts 4 Service (CAS) 5

ECVAM European Centre for the Validation of Alternative Methods 6

EDX Energy Dispersive X-ray 7

HPLC High performance liquid chromatography 8

ICP-MS Inductively coupled plasma mass spectrometry 9

In vitro test method Biological method that uses organs, tissue sections and tissue 10

cultures, isolated cells and their cultures, cell lines and subcellular 11 fractions, or non-biological method that uses chemical interaction 12

studies, receptor binding studies, etc [Rogiers and Beken 2000] 13

ISO International Organization for Standardization 14

IARC International Agency for Research against Cancer 15

IUPAC A system of chemical nomenclature established by the International 16

Union of Pure and Applied Chemistry (IUPAC) 17

Local effects A Local effect refers to an adverse health effect that takes place at 18 the point or area of contact. The site may be skin, mucous 19

membranes, the respiratory tract, gastrointestinal system, eyes, etc. 20 Absorption does not necessarily occur. 21

Nanomaterial An insoluble or biopersistent and intentionally manufactured material 22 with one or more external dimensions, or an internal structure, on 23

the scale from 1 to 100 nm [Regulation (EC) No 1223/2009] 24

Nanoparticle A nano-object with all three external dimensions in the nanoscale 25

[ISO/TS 27687:2008, Nanotechnologies -- Terminology and 26

definitions for nano]. For the purpose of this assessment the term 27 ‘nanoparticle’ is used to also include other forms of nano-object, such 28

as nano-rods, nano-tubes, etc. 29

NPs Nanoparticles 30

Nanoscale Size range from approximately 1 nm to 100 nm [ISO/TS 80004-31 1:2010, Nanotechnologies -- Vocabulary] 32

OECD Organisation for Economic Co-operation and Development 33

PBS Phosphate buffered saline 34

ROS Reactive Oxygen Species 35

SCCNFP Scientific Committee on Cosmetic products and Non-Food Products 36 intended for consumers 37

SCCP Scientific Committee on Consumer Products 38

SCCS Scientific Committee on Consumer Safety 39

SED Systemic Exposure Dosage 40

SEM Scanning electron microscopy 41

Solubility The terms ‘solubility’ and ‘persistence’ are often used to describe the 42 rate of “degradation”. As such there are a number of definitions of 43

solubility (see SCENIHR Opinion ‘Scientific Basis for the Definition of 44

the Term “Nanomaterial”, 8 December 2010). In the context of this 45 assessment, solubility means disintegration of a nanomaterial in an 46

SCCS/1516/13


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aqueous medium or biological environment into molecular 1 components with the loss of nano features. 2

Systemic effects Systemic effect refers to an adverse health effect that takes place at 3 a location distant from the body's initial point of contact and 4

presupposes absorption has taken place. 5

TEM Transmission electron microscopy 6

TiO2: Titanium Dioxide 7

UV-Vis Ultraviolet-visible spectrophotometry 8

Validated method A standard method for which the relevance and reliability have been 9 established for a particular purpose, usually through an inter-lab 10

comparison, which found uncertainties in the measurements 11

acceptable.. 12

VSSA Volume specific surface area (see Kreyling et al., 2010) 13

XRD: X-ray diffraction 14

15

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